SBC 340 Notes

KENYATTA UNIVERSITY
DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY

SBC 340: FORENSIC TECHNIQUES III
OCTOBER 2021 – JANUARY 2022 SEMESTER I
[1] Introduction
(a) Principles of Crime Scene Investigation
• The key principle underlying crime scene investigation is a concept that has become known as
Locard’s Exchange Principle.
• It states that whenever someone enters or exits an environment, something physical is added
to and removed from the scene. This principle is generally summed up by stating: “Every
contact leaves a trace.”
• The logic behind this principle allows investigators to link suspects to victims, to physical
objects, and to scenes.
• Any evidence that can link a person to the scene is referred to as associative evidence. This
may include items such as fingerprints, blood and bodily fluids, weapons, hair, fibers and the
like.
• This type of evidence answers the question “Who did this?” While associative evidence links
people to the place of the crime, reconstructive evidence allows investigators to gain an
understanding of the actions that took place at the scene.
• A broken window, a blood spatter pattern, bullet paths and shoe prints can all reveal what
actually happened. This type of evidence answers the question, “How did it happen?” To help
establish the linkage of people and things to a scene, the investigator may also collect known
substances, called control samples.
• These can be items such as fibers from carpeting at the scene, glass fragments, soil, vegetation
and other trace evidence.
• If these are found on the suspect’s clothing, in their vehicle or at their residence, it could
provide circumstantial evidence linking the person to the scene.
• For example, police are called to a residential neighborhood where a home invasion and
burglary has just occurred.
• Investigators collect glass fragments from a shattered cabinet door with a distinct pattern
etched into the glass.
• A tip leads investigators to a local man with a known history of burglary. Examination of the
suspect’s clothing yields glass fragments with the same distinct pattern as the smashed cabinet
doors.
• Eliminating people who could not be the perpetrator is also important. Control samples of
fingerprints and DNA are often collected from any person(s) who have access to the scene who
are not considered suspects.
(b) Determining the Value of Evidence

1) It is unique -- If an item is found that helps narrow the possibilities of who might be considered
a suspect, or the manner in which a crime was committed, this evidence would be of use. Is an
impression from a vehicle tire found in the dirt at the scene? The tread impression can be
compared to others to determine the type of tire that was on the car. Is a shoe print left in the
soil? The tread may help to identify the size and type of shoes it came from and the wear pattern
could be used to match it to a specific pair.
2) It has a low probability of occurring by chance -- Considering the mathematical
probabilities will help to determine the odds that a piece of physical evidence found at the
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scene could appear merely by coincidence. If DNA Evidence found at the scene matches a
suspect, the chances are exceedingly low that another person could have left this sample. But
even evidence that has a much higher probability—for instance, a common type of shoeprint
that is left in the soil—is still valuable. When combined with other high probability evidence,
these can help narrow the list of possible parties and build a compelling case.
3) It is inconsistent -- If an item is found that is out of place or inconsistent with the setting, or
is out of character for the victim—for instance if the victim was a non--‐smoker but a cigarette
butt is found at the scene—this could be an important bit of evidence.
4) It is a physical match -- If trace evidence is found on the suspect or in his possession that
matches something at the scene, this makes this item valuable as evidence. For instance, broken
plastic parts or a broken fingernail that can be matched by fracture marks can demonstrate that
two pieces were once a part of the same item.
(c) Samples That May Be Collected At a Crime Scene
• A Wide variety of physical evidence can be collected at a scene that is deemed valuable
(“probative”) for collection and investigation:
1) Biological Evidence (e.g., blood, body fluids, hair and other tissues)
2) Latent Print evidence (e.g., fingerprints, palm prints, foot prints)
3) Footwear and tire track evidence

4) Trace Evidence (e.g., fibers, soil, vegetation, glass fragments)
5) Digital Evidence (e.g., Cell phone records, Internet logs, email messages)
6) Tool and tool mark evidence
7) Drug evidence
8) Firearm evidence
• The type of evidence collected will vary with the type of crime. In the case of a burglary, for
example, it would be common to perform tasks in the order listed below. This will help ensure
that evidence isn’t inadvertently damaged or destroyed:
1) Photograph and document the scene
2) Collect trace materials (especially from probable points of entry)
3) Collect low--‐level DNA Evidence by swabbing areas of likely contact
4) Collect other items that may contain biological evidence
5) Locate and collect latent fingerprints
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TOPIC 2: BIOCHEMICALLY SIGNIFICANT COMPOUNDS

(a) Carbohydrates
• Carbohydrates are chemical compounds that contain carbon and the elements of water:
hydrogen and oxygen. A few also contain nitrogen or sulfur.
• There are two main groups of carbohydrates: sugars and starches.
• Sugars are small, water soluble molecules that taste sweet. Starches are very large, insoluble
molecules.
• Carbohydrates may be monosaccharides, disaccharides, or complex carbohydrates known as
polysaccharides.
1) Monosaccharides
• Simple sugars all have the same general formula Cn(H2O)n. The simplest common sugar found
in animals is glucose (C6H12O6).
• Glucose has two molecular forms: a straight chain and a ring. About 98 percent of the sugar
molecules in a solution are in ring form.
• They are divided into:
• Aldoses. Monosaccharides containing an aldehyde group (RCHO) with two or more hydroxyl
groups e.g., glucose. Glucose, also called dextrose, is the most widely distributed sugar in the
plant and animal kingdoms and it is the sugar present in blood as "blood sugar".
• Ketoses with ketone group (R2CO) and two or more hydroxyl groups e.g., fructose. R
represents an alkyl group. Fructose is called levulose or "fruit sugar".
• Monosaccharides are also categorized according to the number of carbon atoms on the main
chain.
No of C Category Moiety Examples

atoms name
Ketotriose 1,3-Dihydroxyacetone
3 Triose
Aldotriose Glyceraldehyde
Ketotetrose or Erythrulose
4 Tetrose Tetrulose
Aldotetrose Erythrose, Threose
Ketopentose Xylulose, Ribulose
5 Pentose
Aldopentose Arabinose, Ribose, Xylose, Lyxose
Ketohexose Fructose, Sorbose, Tagatose, Psicose
6 Hexose Aldohexose Allose, Altrose, Galactose, Glucose,
Gulose, Idose, Mannose, talose
Ketoheptose Mannohetulose, Sedoheptulose
7 Heptose
Aldoheptose Glucoheptose
Properties of monosaccharides
1. The free aldehyde or ketone group in the open-chain configuration of monosaccharides can
reduce cupric ions (Cu2+) to cuprous ions (Cu+) and hence such a monosaccharide is called a
reducing sugar. This is the basis of the Fehling’s and Benedict’s tests for reducing sugars.
The reducing end of such a sugar chain is thus the end that bears the aldehyde or ketone group.
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2. Aldehydes and ketones can add a hydroxyl group to the carbonyl group (R2C=O) to form a
covalent bond. If the hydroxyl carrying compound is an alcohol, the product of addition with
aldehyde is termed as hemiacetal. For the ketone, a reaction with alcohol will result in the
product called hemiketal. This is the basis of ring structure formation in sugars.
3. Ring formation. Aldehyde (aldoses) and ketone (ketose) can form hemiacetal with hydroxyl
of carbon on opposite end of chain to have a single ring structure. This is known as cyclization
of sugars. The 6-membered ring of glucose is called pyranose because of its similarity to
pyran. Aldohexoses form 6 member rings while ketohexoses form 5 member rings known as
furanose because of structural similarity to furan. Aldopentoses normally form 5 member
rings. Fructose can form both 5-membered furanose and 6-membered pyranose rings. The C-
2 carbon keto group in the open fructose chain form can react with C-5 carbon hydroxyl to
form an intramolecular hemiketal called Fructofuranose. Well known examples of 5-
membered ring sugars are found in the nucleic acids e.g. Ribofuranose and 2-
deoxyribofuranose.
4. Biomolecules are classified as either Dextrorotatory (Greek word “dextro” means “right”) or
Laevorotatory (Greek word for “Laevo” for “left”) depending on whether they rotate the
plane-polarized light clockwise or counterclockwise from the point of view of the observer.
The extent of the rotation is proportional to the amount of optically active material that is
present.
5. Fischer convention. In this system the configuration of the groups about an asymmetric centre
is related to that of Glyceraldehyde by a convention thus introduced by Emil Fischer in 1891.
The convention for numbering carbon atoms and naming configurations is as follows:
[1] The carbon atoms are numbered from the end of the carbon chain starting with the
aldehyde or ketone group, which is carbon 1 (C-1)
[2] The symbols D and L refer to the configuration of the asymmetric carbon atom furthest
from the aldehyde or ketone group. In D-Glyceraldehyde the hydroxyl group is
positioned to the right and in L-Glyceraldehyde it is positioned to the left as below. NB:
in this case it is a common misconception that D stands for dextrorotatory and L stands
for laevorotatory.
Thus sugars are given D or L designation depending on whether their configuration

penultimate carbon is the same as that of the given Glyceraldehyde enantiomer. An
example is glucose below
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(a) D- and L-glucose; (b) D- and L-fructose
6. Symmetry, asymmetry & chirality

A carbon atom bonded to 4 groups describes a regular tetrahedron whose mirror image is
symmetrical if 4 groups are identical. The only situation in which the mirror image is not
identical is if all 4 groups bonded to the carbon are different. The molecule is then said to be
asymmetric. Chirality means mirror images cannot be superimposed on the original no matter
the rotation.
a) Enantiomer refers to the mirror images of one molecule to the other
b) Stereoisomer refers to a molecule with n asymmetric centers and no internal plane of
symmetry. They are compounds in which the atoms are linked in the same order but differ
in their spatial arrangement. The D and L stereoisomers of sugars refer to the configuration
of the asymmetric carbon atom furthest from the aldehyde or ketone group. The sugar is
said to be a D isomer if the configuration of the atoms bonded to this carbon atom is the
same as for the asymmetric carbon in D-Glyceraldehyde. If the stereoisomer has one
asymmetric center it will have two 2n=21=2 diastereoisomers
c) Diastereoisomers refer to those molecules which share the same configuration at one
carbon atom but have opposite configuration at another carbon atom. They are not
enantiomers because they are not mirror images of each other. Examples of
diastereoisomers are D-Erythose and D-Threose shown below.
D-Erythrose D-Threose
d) Epimers are sugars differing in configuration only at a single asymmetric center. A good
example is D-Glucose and D-Manose which differ in -OH configuration at C-2. Also D-
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glucose and D-galactose are epimers differing only in their configuration at C-4 as shown
below
D-Glucose D-Mannose
7. α and β classification
Five and six carbon sugars (aldoses and ketoses) exist only momentarily as open chains. An
additional asymmetric carbon is created for example when glucose cyclizes. The C-1 carbonyl
carbon atom in the open chain becomes an asymmetric center in the ring form of the sugar.
Conventionally the ring forms pyranose and furanose as depicted according to some Howard
projections. In these projections the carbon atoms in the ring are not explicitly shown thus
confusing
α-D-Glucose β-D-Glucose
If –OH group is above the plane on the asymmetric carbon we have β while if it is below the
plane then we have α molecule designation. This nomenclature applies to furanose except that
the asymmetric carbon atom is the C-2 and NOT the C-1. The asymmetric carbon is called the
anomeric carbon atom and therefore the α and β forms of molecules are called anomers.
Disaccharides and oligosaccharides are also named using the above nomenclature.
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α-D-Glucopyranosyl -(1→2)- β- fructofuranoside
8. “Boat” and “Chair” conformations

Glucose is sometimes illustrated as a "chair form" because it is a more accurate representation
of the bond angles of the molecule. The "boat" form of glucose is unstable. The chair form is
more stable because of less steric hindrance as the axial positions are occupied by hydrogen
atoms.
β-D-Glucose (chair form) β-D-Glucose (boat form)
9. Mutarotation
In water, α – D – Glucose and β –D – Glucose in the ring forms interconvert through the open
chain form to give an equilibrium mixture. This interconversion which can be detected
practically is called mutarotation. An equilibrium mixture of α and β forms of glucose
consists of 36.4% of α anomer and 63.6% of β forms anomer with very little i.e.<1% open
chained form
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2) Disaccharides
• Disaccharides are sugars made by linking together two monosaccharide rings by a
condensation reaction.
• An OH group from each monosaccharide unit reacts together to make water (H 2O) and form
an oxygen bridge between the sugar rings.
• On hydrolysis with acids or enzymes disaccharides gives two molecules of monosaccharides
which can either be same or different.
Sucrose (Glucose-fructose) Lactose (Galactose-glucose) Maltose (Glucose-glucose)

• Hydroxyl (OH) groups at the end of a disaccharide molecule can link with more rings to make
longer chains. However, most sugars have three rings or fewer.
• The most common disaccharide is sucrose which gives D -(+)- glucose and D-(-)- fructose on
hydrolysis.
• Both the monosaccharides i.e. glucose and fructose are connected through glycosidic linkage
between alpha glucose and second carbon of beta fructose.
• Sucrose being dextrorotatory in nature gives dextrorotatory glucose as well as laevorotatory
fructose on hydrolysis.
• The overall mixture is laevorotatory and this is because laevorotation of fructose (-92.4) is
more than the dextrorotation of glucose (+52.5).
• Sucrose is a non-reducing sugar as both the reducing groups of glucose and fructose are
involved in the glycosidic bond formation.
• Maltose: Maltose is also one of the disaccharide which has two α -D-glucose units which is
connected by first carbon of the glucose and also linked to the fourth carbon of the another
glucose unit. In the solution a free aldehyde can be produced at the first carbon of the second
glucose of the solution and it is a reducing sugar as it shows reducing properties.
• Maltose (C12H22O11) is a disaccharide that is a product of starch digestion and is also found in
some germinating seeds. It is formed by two glucose molecules joined together by a glycosidic
(C-O-C) bond.
• Lactose: Commonly it is called as milk sugar as this disaccharide is found in milk. It is made
up of Beta-D-galactose and β-D-glucose. The bond is between first carbon of galactose and
the fourth carbon of glucose .This is also a reducing sugar.
3) Polysaccharides
• Carbohydrates with large numbers of rings in their molecules are called polysaccharides.
• Polysaccharides are used in living things for energy storage and to build structures
4) Energy storage
• Starches are large polysaccharides formed (synthesized) by joining long chains of
monosaccharide units (such as glucose) together. Since starches are insoluble, they form
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granules within a cell and do not upset the water balance of the cell in the way that the same
amount of soluble sugar would.
• When energy is needed, a reaction, called hydrolysis breaks the starch down into its sugar
molecules. These sugar molecules can then be used to provide energy by respiration.
• Animals use the polysaccharide glycogen as a carbohydrate energy storage molecule.
5) Building structures
• Cell walls in plants are made of a polysaccharide called cellulose. A cellulose molecule may
contain thousands of monosaccharide units bonded together.
• The links between the monosaccharide units in cellulose are arranged to produce a flat
molecule that is stronger than a steel fiber. These molecules run through the cell walls of plants
like the steel rods in reinforced concrete.
6) Polysaccharides in animals
• In animals polysaccharides are mainly used for energy storage. In humans up to 10 percent of
the weight of the liver can be glycogen—an instant store of energy that is easier to mobilize
than fat, which is used for long-term energy storage.
• A typical glycogen molecule may contain 300 to 400 glucose units in a branching molecule.
• Glycogen also occurs in yeasts and bacteria.
• Chitin is made of acetylglucosamine, glucose units with an amino group attached. It is
common in shellfish (the edible crab can be 70 percent chitin) where it is an important part of
the shell.
• Chitin is also found in the exoskeleton of insects.
• Chitin is a structural polysaccharide and is not used as an energy store.
7) Polysaccharides in plants
• Plants store starch as granules inside their cells. Roots such as potatoes, cassava and carrots
are often rich in starch, which provides the energy needed for the next generation to develop
before it can produce its own food by photosynthesis.
• Cellulose is a structural polysaccharide and gives the cell wall its strength. Animals cannot
digest cellulose, and so it passes through the gut largely untouched as roughage.
(b) Lipids
• Many of our common substances are lipids, which include fats and oils (triglycerides),
phospholipids, steroids (or sterols), eicosanoids, lipoproteins, waxes and terpenes.
• Lipids are grouped together because they are (mostly) hydrophobic, nonpolar and not soluble
in water.
• Lipids, like carbohydrates, are formed from hydrocarbon backbones.
• Lipids contain carbon, hydrogen and oxygen
• Most lipids have a large proportion of C-H bonds with a very low proportion of oxygen so
lipids are mostly hydrocarbon
• Lipids are energy rich since the C-H bond is high in energy. Lipids, 9 cal/gm, carbohydrate,
4 cal/gm, protein, 4 cal/gm and alcohol, 7 cal/gm.
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1) Lipid Functions
• Fats are important fuel reserve molecules. Fats are energy rich so they provide concentrated
energy, especially for muscle activity including the heart and respiratory system (breathing).
Humans store fat reserves in adipose tissue. Adipose cells have a remarkable ability to swell
and shrink depending on the amount of fat reserves they contain.
• Phospholipids are the primary structural molecules of cell membranes.
• Fats insulate from cold and provide padding for internal organs.
• Many hormones (regulatory chemicals) are steroids.
• Fats in the diet carry essential fat soluble vitamins: A, D, E and K
• Fats in the diet have satiety value – they stay in stomach longer so we feel fuller longer.
• Fats provide protective coatings on the body surfaces to help prevent dehydration.
2) What are the major categories of lipids found in humans?
1. Triglycerides commonly called fats/oils
2. Phospholipids
3. Sterols
4. Lipoproteins (composite of lipid and protein)
5. Fat-soluble vitamins
[1] Triglycerides: The Fats and Oils (95% of the lipids in our diet)
• First, the terms fats and oils are terms of convention. Fats are "hard" or solid at room
temperature. Oils are liquids at room temperature. All triglycerides have a common structure.
• One molecule of the alcohol, glycerol.
• Attached to the glycerol (by dehydration synthesis or condensation, a reaction that removes
a water molecule from the reacting substances) are 3 fatty acids.
• Fatty acids are chains of hydrocarbons with the carboxyl (acid) functional group (COOH) at
one end of the chain and a methyl group (CH3) at the opposite end.
• The fatty acid hydrocarbon tails are strictly non-polar, so that triglycerides are hydrophobic
molecules.
• The fatty acids determine the characteristics or properties of the fat.
Fatty Acid Differences
1. Length of the Carbon Chain of the Fatty Acids
• Fatty acids have an even number of carbons from 4 – 26 carbon atoms in their hydrocarbon
chain. Most are 14 – 18 carbons in length.
• Short chains are more soluble.
• Short chains are more easily broken down.
• Short chains are less dense.
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• Short chains oxidize more easily (process by which fats become "rancid").
• Butyric acid (in butter) is the shortest fatty acid (4 C)
2. Saturation of the Fatty acid
• Each carbon within the chain has a maximum of 2 places for bonds with hydrogen.
(Remember carbon makes four bonds, two of which are to the adjacent carbon atoms on
the chain).
• If each carbon has 2 hydrogen atoms attached to it, the fatty acid is saturated.
• If two carbon atoms in the fatty acid chain are double bonded to each other, so that there
is less hydrogen in the fatty acid, it is monounsaturated.
• If more than 2 carbon atoms are unsaturated, the fatty acid is polyunsaturated.
• Most plant fats tend to be unsaturated, but fats from tropical plants tend to be very saturated
• Fish oils tend to be unsaturated (from cold water and salt water fish). Other animal fats
tend to be saturated
• Trans-Fatty Acids: The hydrogen atoms attached to the double-bonded carbons are both
on one side (opposite sides) of the carbon chain (either top or bottom). When fats are
hydrogenated, trans-fatty acids tend to form.
• A trans-fatty acid might look like this:
3. Liquid or Solid State at Room Temperatures

• Very short chain fatty acids and unsaturated fatty acids are liquid at room temperature.
The molecules are less dense when they are smaller. Double bonds distort the molecules
so they don't fit close together, which also makes them less dense.
• Saturated chains are solid (denser) because their fatty acid chains fit together better.
4. The stability and spoilage of fatty acids/fats
• Unsaturated fats oxidize more readily, so they have a shorter shelf life, an important issue
with food spoilage.
• Unsaturated and short chain fats are more easily absorbed.
5. The Essential Fatty Acids
• Two groups of polyunsaturated fatty acids are essential: The omega-3 (ω−3) and omega-6
(ω-6) fatty acids.
• At least 3% of the fatty acids we consume should be the essential fatty acids, and ideally,
in about equal proportion. We tend to get more than sufficient omega-6 fatty acids, but
not omega-3 fatty acids.
• Omega (ω) refers to the position of the first double bond of the carbon chain from the
methyl (CH3) end (non-acid end) of the fatty acid. It's a chemistry notation.
Some of the omega (ω ) fatty acids
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Fatty acid Structure Source

Linoleic ω-6 with 18 carbons and 2 double Plant oils
bonds
Arachidonic ω-6 with 20 carbons and 4 double Plant oils
bonds
Linolenic ω-3 with 18 carbons and 3 double seed oils (flax, linseed, walnut,
bonds etc.)
Eicosapentaenoic ω-3 with 20 carbons and 5 double Cold water fatty fish
(EPA) bonds
Docosahexaenoic ω-3 with 22 carbons and 6 double Cold water fatty fish
(DHA) bonds
• Linoleic acid (an omega-6) and Linolenic acid (an omega-3) are not converted or manufactured
from other fatty acids.
• The 20-carbon omega fatty acids are used in the manufacture of eicosanoids, which are a
group of modified fatty acids that are important hormone-like chemical messengers. They
include prostaglandins, thromboxanes and leukotrienes.
• Eicosanoids are important in regulating body functions such as blood pressure, blood-clotting
and immune system responses. Eicosanoids often have antagonistic responses. For example:
1. An ω-6 eicosanoid causes blood clotting and vessel constriction. Its counterpart ω-3
eicosanoid causes blood clotting, but not constriction
2. An ω-6 eicosanoid increases the rate of serum cholesterol breakdown. Its counterpart ω-3
is not as effective; it reduces cholesterol carriers, but doesn't affect breakdown of
cholesterol
[2] Phospholipids
• Phospholipids are modified triglycerides that are the major component of all membranes of
cells. They are also useful emulsifiers, particularly in food products.
• Phospholipids are composed of a glycerol molecule with two fatty acids attached by ester
bonds and a highly polar phosphate-containing compound attached to the third carbon. The
phosphate portion forms the head of the molecule and the two fatty acids form tails.
• Cell membranes are structured from a phospholipid bilayer -- with the hydrophilic heads
pointed to the external and the internal environments, and the hydrophobic fatty acid tails
pointing towards each other.
• The most common phospholipid is lecithin, in which the molecule, choline, is attached to the
phosphate portion. (Choline is sometimes considered a vitamin {B complex}. Choline is also
a component of the neurotransmitter chemical, acetylcholine.)
• Phospholipids are not essential nutrients. They can be manufactured by the body (liver) from
virtually any fats eaten.
• All non-processed food has phospholipid as a part of the cell structure. We digest ingested
phospholipid to: glycerol, fatty acids and phosphate compounds
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[3] Sterols
• Sterols or Steroids are used for a variety of purposes in the body.
1. Vitamins A & D are steroids.
2. Several hormones, including the sex hormones, some growth hormones, and hormones of
the adrenal cortex are steroids.
• Cholesterol, the most abundant steroid, is a structural component of cell membranes, most
abundant in nerve and brain tissue.
• Cholesterol is the precursor for vitamin D and the steroid hormones.
• Cholesterol is a component of bile salts.
• All cholesterol needed is made in the liver from digested fatty acids.
• Dietary cholesterol is not necessarily related to blood levels of cholesterol. The liver
synthesizes 1-3 grams/day vs. 300mg -1 gram ingested.
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• Cholesterol carried by LDL (low density lipoproteins) in the circulatory system is a definite
risk factor for cardiovascular disease, especially artherosclerosis. However it's important to
minimize the amount of cholesterol in circulation, and diet has an important role in doing so.
[4] The Lipoproteins:
1. Chylomicrons
• Very low proportion of protein to lipid
• Carry digested lipids from intestine into lymph and ultimately through the circulatory system
to the liver for processing and repackaging.
2. Very Low-density lipoprotein (LDL)
• Low proportion of protein to lipid.
• Carry triglycerides, cholesterol and other lipids processed and synthesized in the liver to cells
and tissues.
• As VLDL circulate, they release their lipids, and also pick up cholesterol from the body
forming low density lipoproteins
3. Low density lipoprotein (LDL)
• Continue in circulation making their contents available to cells, eventually returning to the
liver for reprocessing. LDLs are proportionally high in cholesterol relative to other lipids.
• A high proportion of LDL is a risk factor in cardiovascular disease
4. High-density lipoprotein (HDL)
• High proportion of protein to lipid
• Carry cholesterol, glycerol and fatty acids released from tissues (especially adipose stores)
back to the liver for processing, recycling or disposal.
(c) Proteins
• Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of
amino acid residues.
• Proteins perform a vast array of functions within organisms, including catalysing metabolic
reactions, DNA replication, responding to stimuli, and transporting molecules from one
location to another.
[1] Amino acids
• Amino acid molecules are made of four groups bonded with a single carbon atom. Three of
these groups are non-variable.
• The amino group NH2 is a basic group, which means it behaves as an alkali in solution.
• At the other end of the molecule is a carboxyl group (COOH), which acts as an organic acid.
• The third group is a hydrogen atom.
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• The fourth group is variable. It is often shown in diagrams by the letter R. Different amino
acids have different R groups.
• There are about 20 naturally occurring amino acids.
• The simplest amino acid is glycine. The R group here is a single hydrogen atom.
• More complicated amino acids, such as proline, have R groups containing many atoms,
complex rings, and sometimes elements such as sulfur or phosphorus.
• Amino acids can join to make chains called polypeptides when the acid group from one amino
acid reacts with the carboxyl group of another. The reaction releases water and produces a link
called a peptide bond.
• More amino acids can be added at each end of the new molecule.
[2] Protein structure
• All proteins are made of small amino acid molecules linked by peptide bonds in long chains
resembling a string of beads.
• The number and order of amino acids in the chain decides how the protein will behave. Some
proteins have more than one chain of amino acids and some have extra groups of atoms added.
• For example, hemoglobin, which transports oxygen from the lungs to cells throughout the
body, is a protein with four amino acid chains wrapped around a central group containing iron.
• Insulin (right) is a small protein molecule with only 51 amino acids on two chains tethered
together by 3 disulfide bridges.
• Some of the large immunity proteins have thousands of amino acids and are bigger than some
simple living organisms!
• The amino acid chain twists as it grows. The twisted chain then forms a spiral. The spiral shape
is held together by links along its length.
[3] Classification of proteins

• There are two main groups of proteins: fibrous and globular.
• Both groups have the same basic structure—they are long chains of amino acids joined by
peptide bonds.
• The difference between the two groups depends on the way the protein chains are arranged.
1) Fibrous proteins
• Fibrous proteins have chains twisted into spiral shapes held together by strong bonds to make
the molecule look like a spring.
• Fibrous proteins can be divided into structural and contractile proteins. Structural proteins form
the structure of an organism. For example, they can be found in skin and hair. Collagen fibers
in the skin give it elasticity and keep it smooth. Contractile proteins such as myosin help
muscles contract.
2) Globular proteins
• Globular proteins have chains that wind in and out of each other, twisting into complex shapes
that look like a ball of wood. Their chains are held together with a mixture of strong and weak
bonds.
• Globular proteins often have more than one chain and can contain extra non-protein groups.
For example, hemoglobin contain iron ions.
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• Globular proteins are often delicate and easily damaged by heat or chemicals. If their molecular
shape is changed by heat they cannot work properly.
• There are various types of globular proteins. Some transport smaller molecules. Some act as
enzymes, controlling the rate of chemical reactions. Some have a protective function. Still
others are hormones, the chemical messengers of the body.
(d) Nucleic acids

• Nucleic acids are macromolecules that store information and provide the instructions for
building proteins. The name nucleic comes from the fact that DNA is found in the nuclei of
eukaryotic cells.
• There are actually two types of nucleic acids: DNA (which stands for deoxyribonucleic acid)
and RNA (for ribonucleic acid).
• The genetic material that humans and other organisms inherit from their parents consists of
giant molecules of DNA.
• The DNA resides in the cell as one or more very long fibers called chromosomes. A gene is a
specific stretch of DNA that programs the amino acid sequence of a polypeptide.
• Those programmed instructions, however, are written in a kind of chemical code that must be
translated from “nucleic acid language” to “protein language”. A cell’s RNA molecules help
make this translation.
• Nucleic acids are polymers made from monomers called nucleotides. Each nucleotide contains
three parts. At the center of each nucleotide is a five-carbon sugar, deoxyribose in DNA and
ribose in RNA.
• Attached to the sugar is a negatively charged phosphate group containing a phosphorus atom
bonded to four oxygen atoms (PO4).
• Also attached to the sugar is a nitrogen containing base (green) made of one or two rings. The
sugar and phosphate are the same in all nucleotides; only the base varies.
• Each DNA nucleotide has one of four possible nitrogenous bases: adenine (abbreviated A),
guanine (G), cytosine (C), or thymine (T). Thus, all genetic information is written in a four-
letter alphabet.
• Dehydration reactions link nucleotide monomers into long chains called polynucleotides. In
the case of DNA, these are called DNA strands. Nucleotides are joined by covalent bonds
between the sugar of one nucleotide and the phosphate of the next.
• This results in a sugar-phosphate backbone, a repeating pattern of sugar-phosphate-sugar-
phosphate, with the bases (A, T, C, or G) hanging off the backbone like appendages. With
different combinations of the four bases, the number of possible polynucleotide sequences is
vast.
• One long polynucleotide may contain many genes, each a specific series of hundreds or
thousands of nucleotides. And each gene stores information in its unique sequence of
nucleotide bases. This sequence is a code that provides instructions for building a specific
polypeptide from amino acids.
• A molecule of cellular DNA is double-stranded, with two polynucleotide strands wrapped
around each other to form a double helix. In the central core of the helix, the bases along one
DNA strand hydrogen bond to bases along the other strand.
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• The bonds are individually weak—they are hydrogen bonds, like those between water
molecules—but collectively they zip the two strands together into a very stable double helix.
• Because of the way the functional groups hang off the bases, the base pairing in a DNA double
helix is specific.
• The base A can pair only with T, and G can pair only with C. Thus, if you know the sequence
of bases along one DNA strand, you also know the sequence along the complementary strand
in the double helix.
• This unique base pairing is the basis of DNA’s ability to act as the molecule of inheritance.
There are many similarities between DNA and RNA.
• Both are polymers of nucleotides, for example, and both are made of nucleotides consisting of
a sugar, a phosphate, and a base. But there are three important differences.
• A first difference: as its name ribonucleic acid denotes, its sugar is ribose rather than
deoxyribose. A second difference between RNA and DNA is that instead of the base thymine,
RNA has a similar but distinct base called uracil (U).
• Except for the presence of ribose and uracil, an RNA polynucleotide chain is identical to a
DNA polynucleotide chain.
• The third difference is that RNA is usually found in single-stranded form, whereas DNA
usually exists as a double helix.
TOPIC 3: NUCLEIC ACIDS ISOLATION, PURIFICATION, IDENTIFICATION AND
QUANTITATIVE ESTIMATION
[1] Isolation of DNA
• Each organism’s genome contains a large amount of DNA that is a potential target for DNA
profiling. DNA is the ‘blueprint of life’, containing all the information that an organism
requires in order to function and reproduce.
• DNA normally exists as a double stranded molecule which adopts a helical arrangement – first
described by Watson and Crick in 1953. Each base is attracted to its complementary base:
adenine always pairs with thymine and cytosine always pairs with guanine
• Human beings contain approximately 3.2b base pairs (bp) of information, which is organized
into a set of 23 chromosomes. Humans contain two sets of chromosomes which add up to a
total of 46 chromosomes – one version of each chromosome inherited from each parent.
• To achieve the highly ordered chromosome structure, the DNA molecule is associated with
histone proteins, which help the packaging and organization of the DNA into the ordered
chromosome structure.
• The use of DNA for analysis or manipulation usually requires that it is isolated and purified to
a certain extent. Specimens must be collected from the environment and given any pre-
treatment necessary to remove exogenous contamination prior to further isolation.
• DNA is recovered from cells by the gentlest possible method of cell rupture to prevent the
DNA from fragmenting by mechanical shearing.
• Cellular proteins that package and protect DNA in the environment of the cell can inhibit the
ability to analyze the DNA and must be separated.
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• The overall goal of an efficient DNA extraction protocol is to remove inhibitors that reduce or
prevent polymerase chain reaction (PCR) amplification and to produce a stable high-quality
DNA that will not degrade over time during storage.
• DNA extraction process typically must:
1) Lyse cells to release the DNA molecules,
2) Separate the DNA molecules from other cellular material,
3) Isolate the DNA into a format compatible with PCR amplification and downstream
applications.
A. Lysis or Cell disruption
• Cell lyses is done in the presence of EDTA which chelates, or binds up, divalent cations such
as calcium (Ca2+) and free magnesium (Mg2+) ions needed by nucleases enzymes (or DNases)
that degrade DNA.
• DNases are protein enzymes found in cells that degrade DNA to allow the cells to recycle
nucleotide components.
• Ideally, cell walls, if present, should be digested enzymatically (e.g. lysozyme treatment of
bacteria), and the cell membrane should be solubilised using a detergent.
• If physical disruption is necessary, it should be kept to a minimum, and should involve cutting
or squashing of cells, rather than the use of shear forces.
• Cell disruption (and most subsequent steps) should be performed at 4°C, using glassware and
solutions that have been autoclaved to destroy DNase activity.
• Autoclaving generally involves heating in saturated steam under a pressure of approximately
15 psi, to achieve a chamber temperature of at least 121°C (250°F) for 15-20 minutes.
• After release of nucleic acids from the cells, RNA can be removed by treatment with
ribonuclease (RNase) that has been heat-treated to inactivate any DNase contaminants.
• RNase is relatively stable to heat as a result of its disulphide bonds, which ensure rapid
renaturation of the molecule on cooling.
B. Separation of DNA molecules
• Proteins and the other major contaminants are removed by shaking the solution gently with
water-saturated phenol, or with a phenol/chloroform mixture, either of which will denature
proteins but not nucleic acids.
• Centrifugation of the emulsion formed by this mixing produces a lower, organic phase,
separated from the upper, aqueous phase by an interface of denatured protein.
• The aqueous solution is recovered and deproteinised repeatedly, until no more material is seen
at the interface.
C. Isolation of DNA for downstream applications
• The deproteinised DNA preparation is mixed with two volumes of absolute ethanol, and the
DNA allowed to precipitate out of solution in a freezer.
• After centrifugation, the DNA pellet is redissolved in a buffer containing EDTA to inactivate
any DNases present. This solution can be stored at 4°C for at least a month.
• Extracted DNA is typically stored at –20°C, or even –80°C for long-term storage, to prevent
nuclease activity.
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• DNA solutions can be stored frozen although repeated freezing and thawing tends to damage
long DNA molecules by shearing. The procedure described above is suitable for total
cellular/genomic DNA isolation.
• If the DNA from a specific organelle or viral particle is needed, it is best to isolate the organelle
or virus before extracting its DNA, since the recovery of a particular type of DNA from a
mixture is usually rather difficult.
• Where a high degree of purity is required DNA may be subjected to density gradient
ultracentrifugation through caesium chloride which is particularly useful for the preparation
of plasmid DNA.
• The quantity and quality of DNA often need to be measured prior to proceeding further with
analytical procedures to ensure optimal results.
• The integrity of the DNA is checked by agarose gel electrophoresis to determine the
concentration of the DNA by using the fact that 1 absorbance unit equates to 50 µgml−1 of
DNA and so:
50×A260=concentration of DNA sample (µgml−1)
• Contaminants may also be identified by scanning UV spectrophotometry from 200 nm to 300

nm. A ratio of 260nm:280nm of approximately 1.8 indicates that the sample is free of protein
contamination, which absorbs strongly at 280 nm. A flow chart of DNA extraction is indicated
in Figure 3.1 below.
Homogenise Cells/Tissues Cellular Lysis Chelating Agents

4℃/sterile equipment Detergent/Lysozyme EDTA/Citrate
Proteinase Agents
Proteinase K
Redissolve DNA Alcohol Precipitation Phenol Extraction

TE Buffer (Tris-EDTA) 70%/100% Ethanol Phenol/Chloroform
Figure 3.1: General steps involved in extracting DNA from cells or tissues.
D. Buffer components
• Extraction buffers for animal DNA, of either modern or ancient origin, typically contain a
buffer (almost universally Tris) to maintain pH, EDTA to chelate divalent cations such as
calcium and magnesium and thereby inhibit DNases, a sodium or potassium salt to stabilize
the nucleic acids in an isotonic medium, and a protease (usually proteinase K) to digest the
proteins.
• An anionic detergent (usually sodium dodecyl sulfate (SDS)) is often included to solubilize
cellular membranes and denature proteins.
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• The buffer pH and concentrations of each component vary—for example, much higher than
normal concentrations of EDTA (500 mM) are used to demineralize bone tissue during
extractions—however, extraction buffers generally maintain the same overall composition.
Undoubtedly, this reflects the relative chemical uniformity of animal cells.
• Unlike animal cells, plant cells commonly contain numerous secondary compounds whose
composition varies from species to species. The cationic detergent
Hexadecyltrimethylammonium bromide (CTAB) is commonly used for solubilizing plant
tissue membranes instead of SDS.
• Both the pH of the isolation buffer and the specific protective agents or detergents included
may need to be optimized for a particular species. The pH should be selected to avoid the
optima of the degradative enzymes.
• For example, most lipolytic enzymes and lipoxygenases have pH optima between 5.0 and 6.0
whereas nuclear DNases have pH optima around 7.0.
• As a result, most plant DNA extraction buffers have a pH of 8.0, though some are as high as
pH 9.0.
• The following components of DNA isolation buffers are used for plant material are aimed at
protecting the DNA from degradation by native enzymes or secondary compounds released
upon disruption of the cells.
• EDTA is usually included to inhibit metal-dependent enzymes by chelating such divalent
cations as Mg2+ and Ca2+; however, at least one EDTA-stimulated DNase is known from wheat.
• Bovine serum albumin is included to cause surface denaturation of degradative enzymes.
• Reducing or sulfhydryl agents such as B-mercaptoethanol, glutathione, cysteine, dithiothreitol
(DTT) and other thiols are commonly included to protect DNA against quinones, disulfides,
peroxidases, and polyphenoloxidases.
• Polyvinylpyrrolidone (PVP) is added to decrease the effect of polyphenols, quinones, and
tannins.
• Less common additives are ascorbic acid as protection against quinones; diethylpyrocarbonate,
bentonite, spermine, spermidine, and other polyamines as protection from RNases;
diethyldithiocarbamate to chelate Cu2+; cyanide as protection against heavy metal oxidases;
NH3 infiltration as protection against H+; ethidium bromide; and coconut milk.
• Aurintricarboxylic acid has also been used to inhibit nucleases in the isolation of plant DNA
and RNA; however, since it protects the nucleic acids from any enzyme action by coating them
it must be removed following the isolation.
• Polysaccharide contaminants either prevent resuspension of the DNA after a precipitation or
prevent digestion with restriction endonucleases.
• One means of dealing with polysaccharide contaminants is to isolate DNA normally, followed
by separation of the DNA from the contaminant using anion exchange chromatography.
• Cheaper and more flexible approaches are either to increase the concentration of NaCl to 2 M
prior to the first alcohol precipitation of DNA or to resuspend the sample in a low ionic strength
buffer, increase the concentration of NaCl to 2 M, and reprecipitate the DNA.
E. Plant samples
• The yield and quality of DNA in plants is greatly affected by the condition of the original tissue
and the means of preserving it prior to extraction.
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• This is particularly true for species that produce large quantities of tannins, phenolics, or other
secondary metabolites that interfere with successful extraction of DNA.
• However, DNA has been successfully isolated from such non-optimal sources as herbarium
sheets and fossils. In general, it is best to harvest the freshest material possible. New growth
early in the season often yields the best results.
• Freshly harvested material should be kept cool and moist (but not submerged), for example, in
an ice chest, until further processing can be undertaken.
• If necessary, subsequent preservation is best done by freezing, although dried specimens may
also be useful sources of DNA.
• It is also important to keep in mind that once frozen, tissues should remain frozen until thawed
in an extraction buffer.
• When adequate refrigeration is not possible, the best means of preserving plant tissue for DNA
analysis appears to be rapid drying in individual bottles containing silicic acid (silica gel Sigma
S7500 and S7625) or anhydrous CaSO4. Tissue dried rapidly (<3 days) is an excellent source
of DNA for years.
• Chemical preservation of plant material generally results in highly degraded DNA; perhaps
due to the inability of the extraction procedure to release DNA from protein-DNA complexes,
rather than due to degradation of the DNA itself.
F. Animal samples
• As with plants, care in the initial collection of animal tissue samples greatly reduces the
problems encountered during DNA isolation.
• Ideally, samples are preserved immediately in ultra-cold freezers or liquid nitrogen, preferably
after dissection into component tissues to enhance both later usefulness and long term
preservation.
• Vertebrate tissues that provide good sources of DNA include blood (particularly the red blood
cells of all vertebrates except mammals and some amphibians, and the buffy coat of
mammalian white blood cells), heart, liver, kidney, stomach, intestine, cerebrospinal fluid, and
skeletal muscle.
• Clotting of blood samples may be prevented with 10 mM EDTA or 0.001% heparin. Blood
cells should be separated from the plasma prior to freezing, and the DNA may be preserved by
diluting samples with sterile 50 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl.
• High molecular weight DNA in animal tissue can be preserved at ambient temperature (for up
to two years in laboratory trials) if cut into 0.5 cm square blocks or smaller, and immersed in
5 vol. or more of 20% dimethylsulfoxide (DMSO) solution (in water), saturated with NaCl.
• Archival tissue samples are also valuable sources of DNA. For example, fresh tissue placed in
10% buffered, neutralized formalin for 3-48 h, dehydrated with xylene, and infiltrated with
liquid paraffin yields DNA for at least five years; in fact, DNA has been analysed from blocks
older than 40 years.
• Not all fixatives are optimal in this application, however; in addition to buffered formalin,
alcohol, acetone, and Omnifix work well.
• Unfavourable tissues for isolation of DNA include those treated with picric acid or mercuric
chloride, or those fixed for long periods in unbuffered or excess formaldehyde.
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• Cryopreservation or formalin-paraffin fixation is often difficult or impossible under field

conditions, so alternative procedures may also be used for sample preservation. One common
means of preserving soft tissue, including plant material, is in alcohol.
• Although isopropanol or propanol may be used, ethanol is preferred because it leads to better
preservation.
• Several problems are particularly acute for ancient or forensic samples. The samples are
generally small and may contain degraded DNA, they may be contaminated by exogenous
DNA (e.g., of bacterial or human origin), and they may yield inhibitory substances co-
purifying with the DNA especially if they were preserved in a sub-optimal chemical or
biological environment.
• Thus, the basic prerequisites for successful extraction of DNA from ancient or forensic samples
are to handle or pre-treat specimens so as to avoid or remove contamination, to use extraction
procedures that do not themselves cause additional DNA degradation, and to remove or to
render inactive inhibitory compounds.
• The following are specific points that are important to take into account in order to minimize
the possibility of contamination while working with ancient or forensic samples; they are
elaborated on in general discussions of the handling of these materials.
(a) Laboratories involved with sample preparation and pre-PCR work should be physically
separated from those involved with PCR and post-PCR analysis.
(b) Laminar flow hoods, sterilized by continuous UV illumination while not in use, should be
used for sample preparation.
(c) Benches and equipment should be cleaned regularly with 5-10% sodium hypochlorite.
(d) Water, reagents (except enzymes, Taq DNA polymerase, and primers), and tubes used for
sample preparation and amplification should be treated with microwave or UV radiation.
Filtration of PCR cocktails (except target DNA and Taq DNA polymerase) through 30000
or 100000 mol. wt cut-off spin columns will also remove contaminating DNA.
(e) A set of pipettes should be dedicated for pre-PCR work, regularly cleaned with 5-10%
sodium hypochlorite, and UV irradiated.
(f) A set of protective clothing (e.g., laboratory coats, face masks, and gloves) should be
dedicated to sample preparation and not other laboratory activities. Gloves should be
changed regularly to prevent cross-contamination.
(g) Extraction controls (i.e., samples lacking tissue but extracted in parallel with those
containing tissue) and amplification controls (i.e., PCRs with water instead of target DNA
template) should be used to monitor contamination.
• Amplification controls containing non-target DNA template are also useful to identify
situations in which the plastics are carriers of contamination.
• A further complication arises with ancient and forensic samples in that different specimens
may have been preserved under very different chemical and biological conditions.
• Some may have been continually frozen in glacial ice, others may have been entombed in
amber, while still others may have been buried in bogs or archaeological sites or preserved in
museums and herbaria.
• Because of this diversity of preservation conditions and because of the importance of the initial
sample preparation steps, e.g., to remove contamination and to prevent further degradation,
distinct procedures are appropriate for different types of samples.
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G. DNA Degradation
• DNA degrades through a variety of mechanisms including both enzymatic and chemical
processes.
• Once an organism dies its DNA molecules face cellular nucleases followed by bacterial,
fungal, and insect onslaughts depending on the environmental conditions.
• The main targets of degradations are:
1) Hydrolytic cleavage at the glycosidic base sugar bond then,
2) Oxidative base damage leading to nucleobase loss and a single-stranded “nick” at the
abasic site.
• Heat and humidity are enemies of intact DNA molecules as they speed up hydrolytic cleavage.
• UV irradiation (e.g., direct sunlight) can lead to crosslinking of adjacent thymine nucleotides
on the DNA molecule which will prevent passage of the DNA polymerase during PCR.
[2] Isolation of RNA

• The methods used for RNA isolation are very similar to those described above for DNA;
however, RNA molecules are relatively short and therefore less easily damaged by shearing,
so cell disruption can be rather more vigorous.
• RNA is, however, very vulnerable to digestion by RNases which are present endogenously in
various concentrations in certain cell types and exogenously on fingers.
• Gloves should therefore be worn, and a strong detergent should be included in the isolation
medium to immediately denature any RNases.
• Subsequent deproteinisation should be particularly rigorous, since RNA is often tightly
associated with proteins. DNase treatment can be used to remove DNA, and RNA can be
precipitated by ethanol.
• One reagent in particular which is commonly used in RNA extraction is guanadinium
thiocyanate which is both a strong inhibitor of RNase and a protein denaturant.
• A flow chart of RNA extraction is indicated in Figure 3.2 below.
Treat Reagents
Treat with RNAase inhibitors Homogenise Cells/Tissues Cellular Lysis
e.g. diethylpyrocarbonate (DEPC) 4℃/treated reagents Detergent/Lysozyme
RNA solvents Proteinase Agents

Guanadinium thiocyanate Proteinase K
Redissolve RNA Alcohol Precipitation Phenol Extraction

70%/100% Ethanol Phenol/Chloroform
Figure 3.2: General steps involved in extracting RNA from cells or tissues.
• It is possible to check the integrity of an RNA extract by analysing it by agarose gel

electrophoresis.
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• The most abundant RNA species, the rRNA molecules 23S and 16S for prokaryotes and 18S
and 28S for eukaryotes, appear as discrete bands on the agarose gel and thus indicate that the
other RNA components are likely to be intact.
• This is usually carried out under denaturing conditions to prevent secondary structure
formation in the RNA. The concentration of the RNA may be estimated by using UV
spectrophotometry.
• At 260 nm 1 absorbance unit equates to 40 µgml−1 of RNA and therefore:
40×A260=concentration of RNA sample (µgml−1)
• Contaminants may also be identified in the same way as that for DNA by scanning UV
spectrophotometry; however, in the case of RNA a 260nm:280nm ratio of approximately 2.0
would be expected for a sample containing no protein.
• In many cases it is desirable to isolate eukaryotic mRNA which constitutes only 2–5% of
cellular RNA from a mixture of total RNA molecules.
• This may be carried out by affinity chromatography on oligo(dT)-cellulose columns. At high
salt concentrations, the mRNA containing poly(A) tails binds to the complementary oligo(dT)
molecules of the affinity column, and so mRNA will be retained; all other RNA molecules can
be washed through the column by further high salt solution.
• Finally, the bound mRNA can be eluted using a low concentration of salt as shown in Figure
3.3 below.
• Nucleic acid species may also be subfractionated by more physical means such as
electrophoretic or chromatographic separations based on differences in nucleic acid fragment
sizes or physicochemical characteristics.
• Nanodrop spectrophotometer systems have also aided the analysis of nucleic acids in recent
years in allowing the full spectrum of information whilst requiring only a very small
(microlitre) sample volume.
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Figure 3.3: Affinity chromatography of poly(A)+RNA.
[3] Techniques used in DNA Extraction

1) Organic (Phenol-Chloroform) Extraction
• Organic extraction involves the serial addition of several chemicals.
• First sodium dodecylsulfate (SDS) and proteinase K are added to break open the cell
membranes and to break down the proteins that protect the DNA molecules while they are in
chromosomes.
• Next a phenol/chloroform mixture is added to separate the proteins from the DNA. The DNA
is more soluble in the aqueous portion of the organic–aqueous mixture.
• When centrifuged, the unwanted proteins and cellular debris are separated away from the
aqueous phase and double-stranded DNA molecules can be cleanly transferred for analysis.
• The method is time-consuming, involves the use of hazardous chemicals, and requires the
sample to be transferred between multiple tubes, which increases the risk of error or
contamination.
2) Chelex Extraction
• Forensic scientists use a chelating-resin suspension that is added directly to the sample (e.g.,
blood, bloodstain, or semen).
• Chelex 100 (Bio-Rad Laboratories, Hercules, CA) is an ion-exchange resin that is added as a
suspension to the samples introduced in 1991 (Walsh et al. 1991).
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• The Chelex method of DNA extraction is more rapid than the organic extraction method. In
addition, Chelex extraction involves fewer steps and thus fewer opportunities for sample
contamination.
• Chelex is composed of styrene divinylbenzene copolymers containing paired iminodiacetate
ions that act as chelating groups in binding polyvalent metal ions such as magnesium.
• The magnesium ions are drawn in and bound by the resin thereby inactivating the DNA-
destroying nuclease enzymes and protecting the DNA molecules.
• In most protocols, biological samples such as bloodstains are added to a 5% Chelex suspension
and boiled for several minutes to break open the cells and release the DNA.
• An initial, prior wash step removes possible contaminants and inhibitors such as heme and
other proteins.
• Exposure to 100°C temperatures denatures the DNA as well as disrupting the cell membranes
and destroying the cell proteins.
• After a quick spin in a centrifuge to pull the Chelex resin and cellular debris to the bottom of
the tube, the supernatant is removed and can be added directly to the PCR amplification
reaction.
• Chelex denatures double-stranded DNA and yields single-stranded DNA from the extraction
process. Thus, it can only be followed by PCR-based analyses.
• The advantage of Chelex extraction is in the removal of inhibitors of PCR and uses only a
single tube for the DNA extraction, which reduces the potential for laboratory-induced
contamination.
• The addition of too much whole blood or too large a bloodstain to the Chelex extraction
solution can result in some PCR inhibition.
3) FTA Paper
• FTA paper was developed by Lee Burgoyne at Flinders University in Australia as a method
for storage of DNA. FTA originally stood for “Fitzco/Flinder Technology Agreement.”
• FTA paper is an absorbent cellulose-based paper that contains four chemical substances to
protect DNA molecules from nuclease degradation and preserve the paper from bacterial
growth. As a result, DNA on FTA paper is stable at room temperature over a period of several
years.
• Use of FTA paper simply involves adding a spot of blood to the paper and allowing the stain
to dry. The cells are lysed upon contact with the paper and DNA from the white blood cells is
immobilized within the matrix of the paper.
• A small punch of the paper is removed from the FTA card bloodstain and placed into a tube
for washing with FTA Purification Reagent (Whatman, Clifton, NJ) to remove heme and other
inhibitors of the PCR reaction.
• This purification of the paper punch can be seen visually because as the paper is washed, the
hemoglobin red color is removed with the supernatant. The clean punch is then added directly
to the PCR reaction.
• Some groups have alternatively performed a Chelex extraction on the FTA paper punch and
used the supernatant in the PCR reaction.
• Devices have also been developed for collection of saliva or buccal cells using a spongy swab
that is then pressed against an FTA card to transfer the collected cells for sample preservation.
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• Indicator paper that changes color with liquid contact is typically used to enable
visualization of sample transfer to the FTA card.
• A major advantage of FTA paper is that consistent results may be obtained without
quantification because a uniform amount of cells are typically sampled.
• The procedure may be automated on a robotic workstation for situations where multiple assays
need to be run.
• A bloodstained punch may be reused for sequential DNA amplifications and typing on the
same sample for multiple assays.
• Due to static electricity, dry paper punches may “jump” between wells in a sample tray. This
method is not as widely used today in forensic analysis as was once envisioned.
• However, due to its preservation and storage capabilities, efforts have been made to use FTA
cards for more widespread collection of DNA samples.
4) Solid-Phase DNA Extraction Methods
• DNA is selectively bound to a substrate such as silica particles. The DNA is retained while
proteins and other cellular components are washed away. Then the DNA is released in a
purified form.
• The most widely used solid-phase extraction methods are QIAGEN columns (QIAGEN, Inc.,
Valencia, CA), DNA IQ (Promega Corporation, Madison, WI), and PrepFiler (Applied
Biosystems).
• The general procedure includes lyses of cells, release of DNA, binding to magnetic beads
containing high concentrations of chaotropic salts such as guanidine hydrochloride, guanidine
isothiocyanate, sodium iodide, and sodium perchlorate, washing the beads, elution and
collection of clean DNA.
• Chaotropic salts disrupt hydrogen-bonding networks in liquid water and thereby make
denatured proteins and nucleic acids more thermodynamically stable than their native folded
or structured counterparts
• Specific DNA extraction methods have been developed for a variety of tissue types including
bloodstains, bone, formalin-fixed paraffin embedded (FFPE) tissues, hair, teeth, urine, and
saliva from stamps and envelopes.
5) Differential Extraction
• Differential extraction is a modified version of the organic extraction method that separates
epithelial and sperm cells.
• Forensic crime laboratories are able to isolate female and male fractions in sexual assault cases
that contain a mixture of male and female DNA.
• The differential extraction procedure involves preferentially breaking open the female
epithelial cells with incubation in an SDS/proteinase K mixture.
• Sperm nuclei are subsequently lysed by treatment with an SDS/proteinase K/dithiothreitol
(DTT) mixture. The DTT breaks down the protein disulfide bridges that make up sperm nuclear
membranes.
• Promega Corporation (Madison, WI) has developed an automated Differex method that
involves using DNA IQ magnetic beads to hold the sperm pellet in place while a separation
solution keeps the digestion buffer and epithelial DNA away from the sperm pellet during the
wash steps.
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• Up to 40 samples can be processed in a 96-well plate in less than 5 hours using Differex
method. However azoospermic semen (i.e., semen without sperm cells as in case of a
vasectomy) cannot be separated from the female fraction with differential extraction.
• In the case of azoospermic perpetrators, the use of Y chromosome specific markers permit
male DNA profiles to be deduced in the presence of excess female DNA.
• Failure to separate the male and female portions of a sexual assault sample results in a mixture
of both the perpetrator’s and the victim’s DNA profiles and such profiles are challenging to
interpret.
6) Laser-capture microdissection (LCM) method
• Laser-capture microdissection (LCM) method captures sperm cells from sexual assault
evidence spread on a microscope slide to perform a reliable STR testing. Laser is an acronym
for Light Amplification by Stimulated Emission of Radiation.
• When sperm cells are observed in the field of view of the microscope, a tiny laser is activated
and a thin plastic film placed over the slide melts at the specific point of laser light contact to
capture or enclose the cell of interest.
• By moving the microscope slide around, dozens of sperm cells are collected onto this thin film
that sits directly above the sample.
• The collection film is then transferred to a tube where DNA from the isolated sperm can be
extracted and amplified using the polymerase chain reaction. Other LCM methods catapult
identified cells directly into a collection tube.
• Non-sperm male cells may also be detected with fluorescent in-situ hybridization (FISH)
techniques enabling selective capture of male versus female cells from a mixed cell population.
7) Direct PCR to Bypass DNA Extraction
• Direct PCR with no DNA extraction is carried out. Improved reaction components and
engineered DNA polymerases permit PCR amplification without DNA extraction and
purification steps to remove inhibitors.
• An advantage of this approach is that samples are effectively concentrated as DNA material is
not lost during wash steps that typically occur with DNA extraction methods.
8) Automated and kit-based extraction of nucleic acids
• Most of the current reagents used in molecular biology and the most common techniques can
now be found in kit form or can be automated, and the extraction of nucleic acids by these
means is no exception.
• The advantage of their use lies in the fact that the reagents are standardised and quality control
tested providing a high degree of reliability.
• For example glass bead preparations for DNA purification have been used increasingly and
with reliable results. Small compact column-type preparations such as QIAGEN columns are
also used extensively in research and in routine DNA analysis.
• Essentially the same reagents for nucleic acid extraction may be used in a format that allows
reliable and automated extraction. This is of particular use where a large number of DNA
extractions are required.
• There are also many kit-based extraction methods for RNA; these in particular have overcome
some of the problems of RNA extraction such as RNase contamination.
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• A number of fully automated nucleic acid extraction machines are now employed in areas
where high throughput is required, e.g. clinical diagnostic laboratories. Here the raw samples
such as blood specimens are placed in 96- or 384-well microtitre plates and these follow a set
computer-controlled processing pattern carried out robotically.
• Thus the samples are rapidly manipulated and extracted in approximately 45 min without any
manual operations being undertaken.
9) Other Methods
a) “Salting out” the DNA with sodium acetate.
b) Use of sodium hydroxide.
c) Digestion of cellular proteins with a thermostable proteinase.
d) A liquid extraction method using the thermostable proteinase approach has been developed
by ZyGEM (Hamilton, New Zealand).
TOPIC 4: PROTEIN ISOLATION AND PURIFICATION METHODS

• Proteins are obtained by isolation procedures depending on the physicochemical properties
of proteins.
• The following characteristics are unique to each protein: amino acid composition, protein
sequence, protein subunit structures, protein size, protein shape, protein net charge,
isoelectric point (pI), protein solubility, protein heat stability and protein hydrophobicity.
• Based on these properties, various methods of isolation exist, e.g. salting out, isoionic
precipitation, etc.
• Proteins purification is quite challenging and, therefore, several approaches are available
e.g. sodium dodecyl sulfate (SDS) gel electrophoresis, chromatography etc.
1) Properties of proteins
(a) Solubility
• Protein solubility properties are summarized as:
1. Forms colloidal solutions in water (due to huge size)
2. Solubility depends on electrostatic charges; net charge depends on number, identity,
location of amino acids and pH of solvent.
3. It depends upon isoelectric point (range 5-8.5): Isoelectric point depends on seven-charged
amino acids, viz. glutamate (δ-carboxyl group), aspartate (β-carboxyl group), cysteine
(thiol group), tyrosine (phenol group), histidine (imidazole side chains), lysine (ε-
ammonium group) and arginine (guanidinium group).
(b) Molecular weight
• Proteins’ molecular weight variation depends on the number of amino acid residues.
• Each amino acid contributes ~110Da value increase in a proteins’ molecular weight, e.g.,
Insulin - 5700 Da; Myoglobin - 1700 Da; Hemoglobin - 64,450 Da.
• Dalton (Da) is an alternate name for the atomic mass unit, and 1 kilodalton (kDa) is 1,000
daltons. Thus a protein with a mass of 64kDa has a molecular weight of 64,000 grams per
mole.
(c) Shape
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• Protein shape varies as globular (insulin), oval (albumin) and fibrous or elongated
(fibrinogen).
• The function of a protein is determined by its shape. The shape of a protein is determined by
its primary structure (sequence of amino acids).
• The sequence of amino acids in a protein is determined by the sequence of nucleotides in the
gene (DNA) encoding it.
(d) Acidic and basic proteins
• Proteins in which the ratio of basic to acidic property is >1, then it is a basic protein and if
the value is <1, then it is an acidic protein.
(e) Color reactions of proteins
• Useful to identify the nature of the amino acids present in proteins e.g., Biuret Test,
Ninhydrin Test, Xanthoproteic Test, Millon's Test, Aldehyde Reaction, Sakaguchi's Test,
Alkali Labile Reaction and Pauly's Test.
(f) In vivo half-life
• This indicates the means time taken by proteins to disappear after its synthesis in cell to its
initial half amount, and is predicted using three model organisms (human, yeast and E. coli).
• “N-end rule”, which relates to the half-life of a protein, is used to identify its N-terminal
residue.
(g) Extinction coefficient
• It indicates the amount of light absorbed by a protein at a certain wavelength. This coefficient
value helps in estimating and identifying a protein when exposed to a spectrophotometer.
• The molar extinction coefficient of a protein can be estimated by knowing its amino acid
composition.
• By using the following equation, the native protein extinction coefficient in water can be
computed using the molar extinction coefficient values at a given wavelength of tyrosine,
tryptophan and cystine (at 280 nm, the extinction value of Tyr is 1490, of Trp is 5500 and of
Cys is 125 in water).
E1 = no. of (Tyr) × Ext (Tyr) + no. of (Trp) × Ext (Trp)

+ no. of (Cystine) × Ext (Cystine)
E2 = no. of (Tyr) × Ext (Tyr) + no. of (Trp) × Ext (Trp)
• Two values of the proteins produced in water at 280 nm using the above equations indicate
that the first value (E1) is due to cysteine residues appearing as half cystines and that the second
value (E2) is due to no cysteine appearing as half cystine.
(h) Aliphatic index
• The aliphatic index of a protein indicates a relative volume occupied by the aliphatic side
chains (alanine, valine, isoleucine and leucine), which also increase the thermostability of the
globular proteins with increasing value.
• This is calculated as below.
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Aliphatic index = {X (Ala) + 𝑎 } × { X (Val) + 𝑏} × {X (Ile) + X (Leu)}
• Where X(Ala), X(Val), X(Ile), and X(Leu) are mole percent (100 × mole fraction) of alanine,
valine, isoleucine, and leucine.
• Where a and b coefficients are the relative volumes of the valine side chain (a = 2.9) and of
the Leu/Ile side chains (b = 3.9) to the side chain of alanine.
(i) GRAVY (GRand AVerage of hydropathicity)
• This is calculated using the Kyte-Doolittle scale as follows:
Sum of AA hydropathy values
GRAVY =
Residue numbers in sequence
• An increasing positive score indicates greater hydrophobicity.

2) Protein sequencing
• The amino acid sequence determination, protein conformation and extent of complexation with
any non-peptide molecules in a protein is called as protein sequencing.
• Methods of protein sequencing are as follows:
1. Edman degradation reaction.
2. Mass spectrometry
(a) Edman degradation reaction

• Ordered amino acid composition of a protein can be determined by using this reaction.
• Peptide sequences up to 50 amino acids long can be sequenced by the Automated Edman
sequencer. The reaction scheme with sequencing steps for protein is as follows:
1. Break any disulfide bridges in protein with an oxidizing agent like performic acid or
reducing agent like 2-mercaptoethanol. Re-forming of disulfide bridges is prevented by
using a protecting group such as iodoacetic acid. Proteins containing more than one chain
are separated and purified
2. Determine the amino acid composition of each chain
3. Determine the terminal amino acid of each chain
4. Break each chain into fragments under 50 amino acids long
5. Separate and purify fragments
6. Determine the sequence of each fragment
7. Repeat with a different pattern of cleavage
8. Construct a sequence of the overall protein.
• Proteins containing 50-70 amino acids cannot be sequenced reliably by the Edman degradation
because long protein chains need to be broken up into small fragments that are sequenced
individually.
• Digestion is performed either by endopeptidases such as trypsin or pepsin or by chemical
reagents such as cyanogen bromide.
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(b) Mass spectrometry

• The protein sequence can be directly determined by this technique using electro-spray
ionization. A protein of any size can be sequenced by this method, but difficulty arises as the
protein size increases.
• Liquid samples for mass spectrometry can be easily prepared due to greater solubility of
peptides as compared with whole proteins.
• In solution, the proteins are digested by an endoprotease and passed through a high-pressure
liquid chromatography column.
• At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high
positive potential into a mass spectrometer.
• The charge on the droplets causes them to fragment until only single ions remain. Peptides are
then fragmented and the mass-to-charge ratio of this is measured.
• This process is repeated with a different digestion enzyme, and overlaps in sequences are used
to construct a sequence for protein.
(c) Determining the amino acid sequences
• Amino acid sequence can be determined by hydrolysis, separation, quantitation.
1. Hydrolysis
• The peptide chain is hydrolyzed into its amino acid using 6M hydrochloric acid (HCl) at 110°C
for 24 h.
2. Separation
• Separation of amino acids from peptides is performed by eluting the mixture of peptide and
buffers (with increasing pH) using a sulfonated polystyrene ion-exchange chromatography
column.
3. Quantitation
• Reactions with ninhydrin quantify the amino acids from a peptide in micrograms. This
reaction results in an intense blue color for most of the amino acids except proline.
• Proline gives a yellow color due to the secondary amino group in its structure. The nanogram
of an amino acid, determined by fluorescamine, is the component that reacts with the alpha-
amino group.
B. Purification of Proteins
• In general, protein purification entails essentially five types of steps:
1) Efficient extraction from biological material;
2) Separation from non-protein components (nucleic acids and lipids);
3) Precipitation steps, initially to recover the bulk protein from a crude extract, followed by
preliminary resolution into manageable fractions;
4) Use of ion-exchange chromatography/size fractionation or hydrophobic chromatography
columns to further separate the target protein-containing fraction from the bulk protein;
5) A more refined set of steps including an “affinity” matrix to enable recovery of the target
protein in a highly purified state along with a high yield.
• A variety of agarose-based matrices with immobilized reactive dyes, covalently bound
nucleotides, metals and numerous other ligands are commercially available (supplied by
Sigma, Amicon, etc).
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• In order to evaluate the progress of purification, a convenient assay procedure—based on

enzymatic activity or some other easily monitored property specific to the protein—should be
available.
• A spectrophotometric or colorimetric method for enzymatic activity measurement is most
convenient and a progressive increase in specific activity (for enzymes, activity in units/mg
protein) is an excellent indicator of the efficacy of the purification step.
• For proteins lacking a readily measurable biological activity, it may be feasible to use an
immunochemical procedure such as western blotting or ELISA (Enzyme-Linked-Immuno-
Sorbent Assay), provided suitable antibodies are available. In this case, electrophoretic
resolution of the protein population in samples at each stage of purification will be required.
• Various methods, listed below, can be used for separation of cellular proteins:
Procedure: Separation Method Property

1. Precipitation
Ammonium sulfate Solubility
Polyethylene glycol Solubility
2. Chromatography
Ion-exchange (anion or cation) Net charge
Hydrophobic interaction Surface hydrophobicity
Metal affinity Metal-binding sites
Ligand affinity Ligand-binding sites (e.g. NAD, NADP)
Gel filtration Subunit/oligomer size, shape
3. Centrifugation Size, shape
C. Characterization of the isolated proteins

• Characterization of proteins can be performed by mass spectrometry/liquid chromatography-
mass spectrometry (LC-MS).
• Prior purification is required to characterize proteins, which can be done by a separation
mechanism or by chromatographic techniques.
• Depending on the properties like diameter, electric charge, hydrophobic domains, group and
biospecific interactions, a particular technique can be selected.
• These are summarized in Table shown below.
Property of Proteins Technique

1 Diameter of molecule Size exclusion chromatography
2 Electric charge of molecule Ion-exchange chromatography
3 Presence of hydrophobic domains Reversed phase chromatography
4 Presence of hydrophobic groups Hydrophobic interaction chromatography
5 Biospecific interaction Affinity chromatography
(a) Introduction to chromatography

• Proteins and other biomolecules can be purified in active form according to solubility, size,
charge, and specific binding affinity.
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• Chromatography works on the principle that different compounds will have different
solubilities and adsorption to the two phases between which they are to be partitioned.
• This process involves: a suitable adsorbent (the stationary phase), solvents or solvent mixtures
(the mobile phase or eluent), and the sample molecules.
• Usually, biomolecule mixtures are subjected to a series of separations, each based on a different
property to yield a pure molecule.
• At each step in the purification, the preparation is assayed and the biomolecule concentration
is determined.
• Substantial quantities of purified proteins, of the order of many milligrams, are needed to fully
elucidate their three-dimensional structures and their mechanisms of action.
• Thus, the overall yield is an important feature of a purification scheme. A variety of
purification techniques are available.
(b) Chromatographic Principles
1. Retention
• Retention or elution volume is the quantity of the mobile phase required to pull the sample
through the column.
• Retention time is defined as how long a component is retained in the column by the stationary
phase relative to the time it resides in the mobile phase.
• The retention is best described as a column capacity ratio (k´), which can be used to evaluate
the efficiency of columns.
• The longer a component is retained by the column, the greater is the capacity factor. The
column capacity ratio of a compound (A) is defined by the following equation:
TA − T0 VA − V0
k′ = =
T0 V0
• Where VA is the elution volume of component A and V0 is the elution volume of a nonretained
compound. At constant flow rate, retention times (T A and T0) can be used instead of retention
or elution volumes.
• Retention factor (Rf): The retention factor (Rƒ) may be defined as the ratio of the distance
traveled by the substance to the distance traveled by the solvent.
• Rƒ values are usually expressed as a fraction of two decimal places. If Rƒ value of a solution is
zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the
solute has no affinity for the stationary phase and travels with the solvent front.
• To calculate the Rƒ value, take the distance traveled by the substance divided by the distance
traveled by the solvent (as mentioned earlier in terms of ratios).
• For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, (2.1/2.8) the
Rƒ value = 0.75.
• Rƒ value depends on temperature and the solvent used in experiment, so several solvents offer
several Rƒ values for the same mixture of compound.
2. Resolution
• Resolution is the ability of the column to separate peaks on the chromatograph.
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• Resolution (R) is expressed as the ratio of the distance between two peak maxima to the mean
value of the peak width at the base line
(TB − TA )2
R=
wA − wB
• Where TB is the retention time of component B, TA is the retention time of component A, wA

is the peak width of component A and wB is the peak width of component B.
• If R is equal to or more than 1, then components are completely separated, but if R is less than
1, then components overlap.
3. Sensitivity
• Sensitivity is a measure of the smallest detectable level of a component in a chromatographic
separation and is dependent on the signal-to-noise ratio in a given detector.
• Sensitivity can be increased by derivatization of the compound of interest, optimization of
chromatographic system or miniaturization of the system.
(c) Techniques in chromatography
(a) Paper chromatography

• Paper chromatography is an analytical method that is used to separate coloured chemicals or
substances, especially pigments. This can also be used in secondary or primary colours in ink
experiments.
• This method has been largely replaced by thin layer chromatography as they involve the same
principles, but is still a powerful teaching tool.
• Double-way paper chromatography, also called two-dimensional chromatography, involves
using two solvents and rotating the paper 90° in between.
• This is useful for separating complex mixtures of compounds having similar polarity, for
example, amino acids.
• If a filter paper is used, it should be of a high quality paper. The mobile phase is developing
solutions that can travel up to the stationary phase carrying the sample along with it.
• Components of the sample will separate readily according to how strongly they adsorb onto
the stationary phase versus how readily they dissolve in the mobile phase.
• When a coloured chemical sample is placed on a filter paper, the colours separate from the
sample by placing one end of the paper in a solvent.
• The solvent diffuses up the paper, dissolving the various molecules in the sample according to
the polarities of the molecules and the solvent.
• If the sample contains more than one colour, that means it must have more than one kind of
molecule.
• Because of the different chemical structures of each kind of molecule, the chances are very
high that each molecule will have at least a slightly different polarity, giving each molecule a
different solubility in the solvent.
• The unequal solubilities cause the various colour molecules to leave solution at different places
as the solvent continues to move up the paper.
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• The more soluble a molecule is, the higher it will migrate up the paper. If a chemical is very
nonpolar it will not dissolve at all in a very polar solvent. This is the same for a very polar
chemical and a very nonpolar solvent.
• It is very important to note that when using water (a very polar substance) as a solvent, the
more polar the colour, the higher it will rise on the paper.
(b) Column chromatography
• In column chromatography, one phase is maintained stationary (the stationary phase) while the
other (the mobile phase) may flow freely over it. We can express the concentration ratio in
such a system as the partition coefficient, K:
𝐶𝑠
𝐾=
𝐶𝑚
• Where Cs and Cm are the sample concentrations in the stationary and mobile phases,
respectively.
• When a mixture made up of several components is applied to such a two phase system, each
component will have its own individual partition coefficient.
• As a result, each will interact slightly differently with the stationary phase and, because of
different partitioning between phases, they will migrate through the column at different rates.
• Since K will be directly affected by the precise experimental conditions (e.g. temperature,
solvent polarity) the chromatographer may vary these to optimize separation.
• In column chromatography, we therefore exploit what are often tiny differences in the
partitioning behaviour of sample molecules to achieve their efficient separation.
(c) Liquid Chromatography
• Liquid chromatography is a separation technique that involves the placement (injection) of a
small volume of liquid sample into a tube packed with porous particles (stationary phase)
where individual components of the sample are transported along the packed tube (column) by
a liquid moved by gravity.
• The components of the sample are separated from one another by the column packing that
involves various chemical and/or physical interactions between their molecules and the
packing particles.
• The separated components are collected at the exit of this column and identified by an external
measurement technique, such as a spectrophotometer that measures the intensity of the color,
or by another device that can measure their amount.
• Note: The modern form of liquid chromatography is now referred to as “flash
chromatography”
(d) Thin Layer chromatography (TLC)
• Thin Layer Chromatography is a chromatography in which compounds are separated on a thin
layer of adsorbent material, typically a coating of silica gel on a glass plate or plastic sheet.
• TLC is a solid-liquid technique in which the two phases are a solid (stationary phase) and a
liquid (moving phase).
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• Solids most commonly used in chromatography are finely divided silica gel (SiO 2 × H2O) and
alumina (Al2O3 × H2O). Both of these adsorbents are polar, but alumina is more so. Silica is
also acidic. Alumina is available in neutral, basic, or acidic forms.
• Thin Layer Chromatography (TLC) is a sensitive, fast, simple and inexpensive analytical
technique. It is a micro technique; as little as 10-9g of material can be detected, although the
sample size is from 1 to 100×10-6g.
• TLC involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that
is coated with a thin layer of an adsorbent.
• The sheet, which can be the size of a microscope slide, is placed on end in a covered jar
containing a shallow layer of solvent.
• As the solvent rises by capillary action up through the adsorbent, differential partitioning
occurs between the components of the mixture dissolved in the solvent the stationary adsorbent
phase.
• The more strongly a given component of a mixture is adsorbed onto the stationary phase, the
less time it will spend in the mobile phase and the more slowly it will migrate up the plate.
Diagrammatic representation of TLC
• The following are some common uses of Thin-Layer Chromatography:

1. To determine the number of components in a mixture.
2. To determine the identity of two substances.
3. To monitor the progress of a reaction.
4. To determine the effectiveness of a purification.
5. To determine the appropriate conditions for a column chromatographic separation.
6. To monitor column chromatography
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• Today TLC has gained increasing importance as an analytical separation technique, which is
probably due to effects of instrumentation and automation
• For instance, high performance thin layer chromatography (HPTLC), also called planar
chromatography, is based on a multistage distribution process involving suitable adsorbents
(the stationary phase) coated as a thin layer onto a suitable support (e.g., glass plate, polyester
or aluminium sheet), solvents or solvent mixtures (the mobile phase or eluent) and sample
molecules
(e) Salting Out
• Most proteins are less soluble at high salt concentrations, an effect called salting out. The salt
concentration at which a protein precipitates differs from one protein to another. Hence, salting
out can be used to fractionate proteins.
• For example, 0.8 M ammonium sulfate precipitates fibrinogen, a blood-clotting protein,
whereas a concentration of 2.4 M is needed to precipitate serum albumin.
• Salting out is also useful for concentrating dilute solutions of proteins, including active
fractions obtained from other purification steps.
(f) Dialysis
• Proteins can be separated from small molecules by dialysis through a semipermeable
membrane, such as a cellulose membrane with pores.
• Molecules having dimensions significantly greater than the pore diameter are retained inside
the dialysis bag, whereas smaller molecules and ions traverse the pores of such a membrane
and emerge in the dialysate outside the bag.
• This technique is useful for removing a salt or other small molecule, but it will not distinguish
between proteins effectively.
(g) Gel-Filtration Chromatography
• The sample is applied to the top of a column consisting of porous beads made of an insoluble
but highly hydrated polymer such as dextran or agarose (which are carbohydrates) or
polyacrylamide.
• Sephadex, Sepharose, and Bio-gel are commonly used commercial preparations of these
beads, which are typically 100 µm (0.1 mm) in diameter.
• Small molecules can enter these beads, but large ones cannot. The result is that small molecules
are distributed in the aqueous solution both inside the beads and between them, whereas large
molecules are located only in the solution between the beads.
• Large molecules flow more rapidly through this column and emerge first because a smaller
volume is accessible to them.
• Molecules that are of a medium size enter a bead and flow from the column at an intermediate
position, and small molecules, which take a longer, tortuous path, will exit last.
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Diagram of a gel-filtration process

(h) Ion-Exchange Chromatography
• A good example of adsorption chromatography is ion exchange chromatography where the
sample components are eluted by means of a gradient of competing salt counterions after all
the sample has been loaded onto and washed completely through the column
• Proteins can be separated on the basis of their net charge by ion-exchange chromatography. If
a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing
carboxylate groups, whereas a negatively charged protein will not
• A positively charged protein bound to such a column can then be eluted (released) by
increasing the concentration of sodium chloride or another salt in the eluting buffer because
sodium ions compete with positively charged groups on the protein for binding to the column.
• Proteins that have a low density of net positive charge will tend to emerge first, followed by
those having a higher charge density.
• Positively charged proteins (cationic proteins) can be separated on negatively charged
carboxymethyl-cellulose (CM-cellulose) columns.
• Conversely, negatively charged proteins (anionic proteins) can be separated by
chromatography on positively charged diethylaminoethyl-cellulose (DEAE-cellulose)
columns.
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(i) Affinity Chromatography

• This technique takes advantage of the high affinity of many proteins for specific chemical
groups.
• For example, the plant protein concanavalin A can be purified by passing a crude extract
through a column of beads containing covalently attached glucose residues.
• Concanavalin A binds to such a column because it has affinity for glucose, whereas most other
proteins do not.
• The bound concanavalin A can then be released from the column by adding a concentrated
solution of glucose.
• The glucose in solution displaces the column-attached glucose residues from binding sites on
concanavalin A
• Affinity chromatography is a powerful means of isolating transcription factors, proteins that
regulate gene expression by binding to specific DNA sequences.
• A protein mixture is percolated through a column containing specific DNA sequences attached
to a matrix; proteins with a high affinity for the sequence will bind and be retained.
• In this instance, the transcription factor is released by washing with a solution containing a
high concentration of salt.
• In general, affinity chromatography can be effectively used to isolate a protein that recognizes
group X by:
(1) Covalently attaching X or a derivative of it to a column,
(2) Adding a mixture of proteins to this column, which is then washed with buffer to remove
unbound proteins, and
(3) Eluting the desired protein by adding a high concentration of a soluble form of X or altering
the conditions to decrease binding affinity.
• Affinity chromatography is most effective when the interaction of the protein and the molecule
that is used as the bait is highly specific.
• This classic form of chromatography is based upon the principle that certain solid materials,
collectively known as adsorbents, have the ability to hold molecules at their surface.
• This adsorption process, which involves weak, non-ionic attractive forces of the van der Waals’
and hydrogen-bonding type, occur at specific adsorption sites.
• These sites have the ability to discriminate between types of molecules and are occupied by
molecules of either the eluent or of the analytes in proportions determined by their relative
strength of interaction.
• As eluent is constantly passed down the column, differences in these binding strengths
eventually lead to the separation of the analytes.
• Silica is a typical adsorbent. It has silanol (Si-OH) groups on its surface, which are slightly
acidic, and can interact with polar functional groups of the analyte or eluent. Other commonly
used adsorbents are alumina and carbon.
(j) Gas Chromatography
• Some molecules of importance in biochemistry may be converted to derivatives that are
structurally stable, though volatile, in the temperature range 200–250°C.
• Good examples are trimethylsilated sterols and carbohydrates (esterified at their hydroxyl
groups), and methylated esters of fatty acids.
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• A second category of molecules (e.g. ethanol) is stable and volatile at somewhat lower
temperatures without derivatization.
• Both of these groups of molecules may be analyzed by gas chromatography (GC, also
sometimes called gas-liquid chromatography; GLC).
• This is a form of partition chromatography in which a volatile sample is carried in an inert gas
mobile phase (the carrier gas) such as nitrogen, helium or argon and applied to a narrow
column (0.1 to 0.5 cm diameter) ranging in length from one to thirty meters that contains a
liquid stationary phase.
• Sample components may be detected using flame ionization, flame photometry or thermal
conductivity detectors.
• If the separated sample components are to be collected for further analysis (or for an interfaced
method such as mass spectrometry), the carrier gas flow may be split before it enters the flame
ionization detector.
• Varying column temperature, gas flow and type of column may optimize GC separations.
(k) High-Pressure Liquid Chromatography
• The resolving power of all of the column techniques can be improved substantially through the
use of a technique called high-pressure liquid chromatography (HPLC), which is an enhanced
version of the column techniques already discussed.
• The column materials themselves are much more finely divided and, as a consequence, there
are more interaction sites and thus greater resolving power.
• Because the column is made of finer material, pressure must be applied to the column to obtain
adequate flow rates. The net result is high resolution as well as rapid separation.
• The high resolution is achieved at the cost of the generation of high pressures, relatively low
flow rates and the constraints the high pressure imposes on the instrumentation.
(l) Electrophoresis
1. Polyacrylamide Gel electrophoresis: Agarose gel electrophoresis is commonly used to
separate DNA and RNA, while polyacrylamide gel electrophoresis is the most common
method for separating proteins. Polyacrylamide gel is the result of polymerizing acrylamide
monomers into long chains and then cross-linking the chains with a bifunctional compound. A
number of bifunctional cross-linkering compounds are known including ethylene diacrylate,
N,N’-bisacrylycystamine (BAC), N,N’-diallyltartardiamide (DATD) and the commonly used
N,N’-methylene bisacrylamide (bis meaning two). Polymerization of acrylamide and
bisacrylamide is catalyzed in the presence of either ammonium persulphate or riboflavin by
the compound N,N,N,N’-tetramethylethyldiamine (TEMED) or, less commonly, 3-
dimethylamino proprionitrile (DMAPN). TEMED catalyzes the formation of free radicals
from persulphate and these free radicals initiate and accelerate polymerization. Polymerization
rates can be increased by increasing TEMED or persulphate concentrations
2. Capillary electrophoresis (CE): Electrophoresis is done in a buffer filled, narrow-bore

capillaries of about 25-100μm in internal diameter and 25-75 cm long with 50 and 75 micron
being the most commonly employed inner diameters. Operation of a CE system involves
application of a high voltage (typically 10-30kV) across a narrow bore (25-100μm) capillary.
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The capillary is filled with electrolyte solution which conducts current through the inside of
the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte.
3. Isoelectric Focusing: Proteins can also be separated electrophoretically on the basis of their
relative contents of acidic and basic residues. The isoelectric point (pl) of a protein is the pH
at which its net charge is zero.
• At this pH, its electrophoretic mobility is zero because z in equation 1 is equal to zero. For
example, the pI of cytochrome c, a highly basic electron-transport protein, is 10.6, whereas
that of serum albumin, an acidic protein in blood, is 4.8.
• Suppose that a mixture of proteins undergoes electrophoresis in a pH gradient in a gel in the
absence of SDS. Each protein will move until it reaches a position in the gel at which the pH
is equal to the pI of the protein.
• This method of separating proteins according to their isoelectric point is called isoelectric
focusing.
• The pH gradient in the gel is formed first by subjecting a mixture of polyampholytes (small
multicharged polymers) having many pI values to electrophoresis.
• Isoelectric focusing can readily resolve proteins that differ in pI by as little as 0.01, which
means that proteins differing by one net charge can be separated
4. Two-Dimensional Electrophoresis: Isoelectric focusing can be combined with SDS-PAGE
to obtain very high resolution separations.
• A single sample is first subjected to isoelectric focusing. This single-lane sample is then placed
horizontally on top of an SDS-polyacrylamide gel slab.
• The proteins are thus spread across the top of the polyacrylamide gel according to how far they
migrated during isoelectric focusing. They then undergo electrophoresis again in a
perpendicular direction (vertically) to yield a two dimensional pattern of spots.
• In such a gel, proteins have been separated in the horizontal direction on the basis of isoelectric
point and in the vertical direction on the basis of mass.
• Therefore, two-dimensional electrophoresis separates proteins of identical molecular weight
that differ in pI, or proteins with similar pI values but different molecular weights.
• It is remarkable that more than a thousand different proteins in the bacterium Escherichia coli
can be resolved in a single experiment by two-dimensional electrophoresis
• It is now possible to identify proteins by coupling two-dimensional gel electrophoresis with
mass spectrometric techniques.
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Isoelectric focusing. This technique separates Two-dimensional electrophoresis.

proteins according to their isoelectric points. A stable Proteins are first separated by isoelectric
pH gradient is established in the gel by the addition focusing in a cylindrical gel. The gel is
of appropriate ampholytes. A protein mixture is then laid horizontally on a second, slab-
placed in a well on the gel. With an applied electric shaped gel, and the proteins are
field, proteins enter the gel and migrate until each separated by SDS polyacrylamide gel
reaches a pH equivalent to its pI. Remember that electrophoresis. Horizontal separation
when pH = pI, the net charge of a protein is zero reflects differences in pI; vertical
separation reflects differences in
molecular weight.
TOPIC 5: PROPERTIES OF ANTIBODIES
• Antibodies belong to a group of proteins called immunoglobulins (Igs) that are present in the
blood of immunized animals. They are generated as a result of an immune response.
• The removal of cells and fibrin from blood is used to collect the serum fraction frequently
referred to as antiserum.
• Listed in order of decreasing quantity found in plasma or serum, immunoglobulins comprise
five major classes: immunoglobulin G (IgG), IgA, IgM, IgD and IgE.
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(a) Antibody Class Functions

1. IgG Antibodies: IgG is the dominant antibody found in blood. Functionally, IgG promotes
the uptake of microorganisms by immune cells and is responsible for long term protection from
disease as a secondary response. For example, this antibody is used in the detection of blood
viruses. Various subclasses can all fix complement (via classical or alternate pathway). Can be
transferred across the placenta into the fetal circulation. Produced late in immune responses.
Opsonizing activity via Fc receptors present on macrophages and PMNs.
2. IgM Antibodies: IgM is the first antibody produced as a primary response to infection and is
very effective in activating complement (complement fixation) and destroying bacteria. It is a
pentamer of the basic (two light chain two heavy chain) structure. The pentamer is held
together by extra disulfide bonds formed near the C-terminal end of the heavy chains plus an
additional short polypeptide referred to as J chain/piece (for “joining”) as does polymeric IgA.
IgM is often used in assays for early detection of infection. They serve as membrane receptors
of B-cells (as “IgMs”, an “IgG-like” monomer.
3. IgA Antibodies: IgA is known as the secretory antibody. It is found in mucous membrane
secretions as a dimer associated with a secretory chain. While IgA is the second-most abundant
serum Ig, it is most characteristically a secretory immunoglobulin, and is the most abundant Ig
in exocrine secretions (saliva, bile, mucus of the gut and respiratory tract, milk, etc.). It
protects the respiratory and gastrointestinal tracts from infection. In some mammals, IgA is a
major factor in milk that passively protects the newborn. IgA in serum is mostly an α2L2
monomer; IgA in secretions is polymeric (mostly dimer, i.e. [α2L2]2, with some trimer),
includes an S-piece (for “secretory”), and like IgM also bears the J-piece.
4. IgE Antibodies: IgE binds to the Fc receptors on mast cells and basophils and triggers the
allergic response. It is also associated with defense against parasites. It is present in blood in
extremely small concentrations. Extremely low levels in serum. Typically, this antibody is
used in allergy testing. While antibodies are generally very stable, IgE is an exception and is
characteristically heat labile.
5. IgD Antibodies: IgD is a minor blood component. It is typically bound to the surface of B-
lymphocytes. Rare in serum; no other known biological function.
Summary of Ig isotypes or classes
Antibody isotype Action
IgA Gut, respiratory, urinary response
IgD Initial immune system response
IgE Response to allergens-histamine release
IgG Immune response to invading pathogens
IgM Early immune response for pathogens
Summary of Immunoglobulins structures

• The chemical structure of antibodies explains three functions of antibodies:
(1) Binding versatility,
(2) Binding specificity, and
(3) Biological activity.
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• Each is composed of two identical heavy chains (H) and two identical light chains (L). The H
chains differ in antigenic and structural properties, and determine the class and subclass of the
molecule.
• The two L chains are either of type kappa (κ) or lambda (λ). Distribution of κ and λ chains
differs in all Ig classes and subclasses, as well as between different species.
• Covalent interchain disulfide bridges join L to H and H to H chains. By participating in the
tertiary structure, they confer greater stability to the immunoglobulin molecule.
• IgG isotype is preferred because its generation and binding is more consistent. IgM antibodies
can be used if no other isotype is available.
• The IgG molecules can be broken down into four subclasses, IgG1, IgG2, IgG3, and IgG4.
(b) Some useful definitions in Antibody-Antigen Interactions -
1. Immunogen - What is injected into an animal to induce an immune response.
2. Antigen - An antigen is a protein, peptide, or molecule used to cause an immune response in
an animal. It is that molecule that an antibody binds with high affinity and specificity. Often
these molecules are large, sometimes they are proteins that are even larger than an antibody
molecule. In fact, in some cases they are not a single molecule, but may be an organism such
as a bacteria or virus.
3. Epitope- What an antigen binding site actually envelops and binds to. The animal responds by
making antibodies to individual epitopes located on the antigen. An epitope can be an amino
acid sequence on a denatured peptide or a several sequences on the surface of a folded protein.
A large protein or an organism may have many epitopes. In some cases, each one is different
in shape. In some cases, the same epitope is repeated several times. Animals frequently
generate multiple antibodies to the same epitope
(c) Properties of antibodies:
1. Antibodies uniquely bind to a protein or other molecule.
2. Antibody binding to molecules is essentially permanent at physiological conditions.
3. New antibodies can be made tailored to new interesting molecules.
4. The basis of antibody-antigen binding is steric complementarity between the combining site
and the epitope or antigenic determinant ("lock and key" concept). A variety of non-covalent
forces are also involved in this binding:
(a) Van der Waals interactions. These are very weak and extremely short-range forces (they
decrease with the seventh power of the distance), but they become important when many
such interactions over the large area of an interactive surface are added together.
(b) Hydrophobic interactions. Exclusion of water molecules between hydrophobic surfaces
can also be a major contributor to antigen-antibody binding.
(c) Hydrogen bonds and ionic interactions can also provide considerable stabilization of
antibody-antigen binding, in those cases where the epitope is capable of participating in
such interactions.
5. Speed. In a “typical” primary response we see a lag of about 4 days, followed by a slow rise
of antibody to a peak at about two weeks. In the secondary, the lag is only two days, and
antibody levels rise very rapidly to a peak within 4-6 days.
6. Antibody levels. The secondary response reaches peak antibody levels which are very much
higher than in the primary.
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7. Duration. Primary responses not only appear more slowly; they disappear fairly rapidly.
Antibody titers in secondary responses remain at high levels for longer periods of time, and in
humans may persist for many years.
8. Ig class. The majority of the antibody in a primary response is IgM, with some IgG appearing
later in the response; in a secondary, IgG is the predominant antibody throughout the response.
IgM also appears during a secondary response, but at levels and with a time course comparable
to the primary and is therefore swamped out by the higher levels of IgG. IgM does not exhibit
immunological memory: it does not appear more rapidly or at higher levels in secondary
responses, and does not undergo significant affinity maturation.
9. Affinity. The average affinity of antibody in a secondary response is higher than in a primary
response. Even during the course of a particular secondary response, it can be shown that the
affinity of "late" antibody is higher than that of "early" antibody. This progressive increase in
the average affinity of antibody is known as Affinity Maturation.
(d) Factors Affecting Antigen Antibody Reactions
• Many factors affect the interaction between antigen and antibody; these include specificity,
cross reactivity, temperature, PH, ionic strength, concentration, and intermolecular specificity.
1) Specificity: The ability of a particular antibody to combine with one antigen instead of another
is referred to as specificity. This property resides in the portion of the antigen binding fragment
of an immunoglobulin molecule. Antigen-antibody reactions can show a high level of
specificity.
2) Cross reactivity: When some of the determinants of an antigen are shared by similar antigenic
determinants on the surface apparently unrelated molecules, a proportion of the antibodies
directed against one kind of antigen will also react with the other kind of antigen. This is called
cross reactivity.
• Antibodies directed against a protein in one species may also react in a detectable manner with
the homologous protein in another species, which is another example of cross reactivity.
• Example of cross reactivity: Three organisms might possess antigenic structures and produce
corresponding antibodies as follows:
Organism Antigens Antibodies

1 ABC abc
2 BCD bcd
3 DE de
• Antiserum prepared from organism 1 will react with organism 1 & 2. Antiserum prepared from
organism 2 will react with 1, 2, & 3. Antiserum produced from organism 3 will react organism
2 & 3 but not with organism 1.
3) Temperature: The optimum temperature needed to reach equilibrium in an antibody-antigen
reaction differs for different antibodies. IgM antibodies are cold reacting with thermal range
4-22°C, and IgG antibodies are warm reacting, with an optimum temperature of reaction at
37°C
4) pH: Although the optimum pH for all reactions has not been determined, a pH of 7.0 is used
for routine laboratory testing.
5) Ionic strength: The concentration of salt in the reaction medium has an effect on antibody
uptake by the membrane bound erythrocyte antigens. Sodium and chloride ions in solution
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have inhibition effect. These ions cluster around and partially neutralize the opposite charges
on antigen and antibody molecules, which hinders the association of antibody with antigen.
Reducing or lowering the ionic strength of a reaction medium such as low-ionic strength salt
can enhance antibody uptake.
6) Concentration: Under normal conditions the concentration of antigen and antibody should be
optimal but sometimes this fails to happen in which excess antibody or antigen concentration
will result in false reaction known as zonal reaction. When the concentration of antigen is
excess it is known as post zone reaction; excess antibody is referred as prozone reaction. This
phenomenon can by overcome by serial dilution until optimum amount of antigen and antibody
will present.
7) Bond strength and inter molecular attractive force: Bonding of an antigen to an antibody
takes place because of the formation of multiple, reversible, intermolecular attraction between
an antigen and amino acids of the binding site. The bonding of antigen to antibody is
exclusively non covalent.
• The attractive force of noncovalent bonds is weak when compared to covalent bonds, but the
formation of multiple non covalent bonds produces considerable total-binding energy.
• The strength of a single antigen- antibody bond is termed antibody affinity. The strongest
bonding develops when antigens and antibodies are close to each other and when the shapes
of both the antigenic determinate and the antigen-binding site conform to each other. This
complementary matching is referred to as goodness of fit.
TOPIC 6: WESTERN BLOT TECHNIQUE
• A western blot is a laboratory method used to detect specific protein molecules from among a
mixture of proteins. This mixture can include all of the proteins associated with a particular
tissue or cell type.
• Western blots can also be used to evaluate the size of a protein of interest, and to measure the
amount of protein expression. This procedure was named for its similarity to the previously
invented method known as the Southern blot.
• The first step in a western blot is to prepare the protein sample by mixing it with a detergent
called sodium dodecyl sulfate, which makes the proteins unfold into linear chains and coats
them with a negative charge.
• Next, the protein molecules are separated according to their sizes using a method called gel
electrophoresis.
• Following separation, the proteins are transferred from the gel onto a blotting membrane.
Although this step is what gives the technique the name "western blotting," the term is typically
used to describe the entire procedure.
• Once the transfer is complete, the membrane carries all of the protein bands originally on the
gel.
• Next, the membrane goes through a treatment called blocking, which prevents any nonspecific
reactions from occurring.
• The membrane is then incubated with an antibody called the primary antibody, which
specifically binds to the protein of interest.
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• Following incubation, any unbound primary antibody is washed away, and the membrane is
incubated yet again, but this time with a secondary antibody that specifically recognizes and
binds to the primary antibody.
• The secondary antibody is linked to a reporter enzyme that produces color or light, which
allows it to be easily detected and imaged. These steps permit a specific protein to be detected
from among a mixture of proteins.
Western blotting work flow

(a) Preparation of Cell Lysates for Western blots:
• Prepare total cell lysates by solubilizing cells in an appropriate sample buffer, such as 2×SDS
sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF
and bromophenyl blue), at approximately 2x106-1x107 cells per mL.
• The extracts are heated in a boiling water bath for 5 minutes and then sonicated with 3-4 bursts
of 5-10 seconds each.
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(b) Immunoblotting:
1) Prepare the following solutions:
Blotting Buffer Blocking Solution Antibody Solution
25 mM Tris, pH 2-5% nonfat dry milk in Blotting 1-5% nonfat dry milk in Blotting
7.4 Buffer Buffer
0.15 M NaCl Adjust pH to 7.4 Adjust pH to 7.4
0.1% Tween® 20
2) Transfer the electrophoresed proteins to a PVDF (Polyvinylidene fluoride) membrane and
incubate for 1 hour at room temperature in Blocking Solution.
3) Incubate the membrane overnight at 4°C in Antibody Solution containing primary antibody.
4) Wash the membrane at room temperature for 30-60 minutes with 5 or more changes of Blotting
Buffer.
5) Incubate the membrane for 1 hour at room temperature in Antibody Solution containing
appropriate dilution of HRP (Horseradish Peroxidase)-conjugated secondary antibody.
6) Wash the membrane for 30-60 minutes with 5 or more changes of Blotting Buffer.
7) Detect with Chemiluminescent Detection Substrate.
8) Expose to film and develop image.
NB: Horseradish Peroxidase (HRP) has a high turnover rate that allows generation of strong
signals in a short time span. HRP secondary antibodies are commonly used in western blot (WB),
immunohistochemistry (IHC) and ELISA due to the small size, stability and lower cost of the
enzyme. The substrates commonly used with HRP fall into different categories including
chromogenic (e.g. DAB, TMB, OPD), fluorogenic (e.g. ADHP) and chemiluminescent (e.g. ECL)
substrates depending on whether they produce a colored, fluorimetric or luminescent derivative
respectively.
(c) Medical diagnostic applications

1) The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human
serum sample. Proteins from known HIV-infected cells are separated and blotted on a
membrane as above. Then, the serum to be tested is applied in the primary antibody incubation
step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme
signal is added. The stained bands then indicate the proteins to which the patient’s serum
contains antibody.
2) A western blot is also used as the definitive test for bovine spongiform encephalopathy (BSE,
commonly referred to as 'mad cow disease').
3) Some forms of Lyme disease testing employ western blotting.
4) A western blot can also be used as a confirmatory test for Hepatitis B infection.
5) In veterinary medicine, a western blot is sometimes used to confirm FIV+ status in cats.
TOPIC 7: ANALYSIS OF FIRE AND EXPLOSIONS

A. Fires
(a) The nature of fire
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• Fire causes considerable damage, loss of life and human misery in the world. Fire is that
condition, characterized by the evolution of heat and light that occurs as a consequence of a
chemical process known as combustion.
• Combustion is an exothermic (i.e. heat-evolving) redox reaction. Redox reactions are those
that involve the complete transfer of electrons from one chemical species to another.
• The chemical species that loses electrons is referred to as the reductant or reducing agent,
whereas the species that gains them is known as the oxidant or oxidizing agent. In combustion
reactions, the reductant is referred to as the fuel.
• In the vast majority of fires, the oxidant is the molecular oxygen (O 2(g)) that makes up 20.95
per cent by volume of dry normal air at sea level. Such fires are referred to as conventional
fires.
• Under unusual circumstances, other oxidants may be involved. For example, fires involving
fireworks are sustained, at least in part, by the oxidising agents (such as the nitrate ion, NO3–)
present within the formulations of the contents of the fireworks themselves.
• Four conditions that must be met in order for a fire to start and to be self-sustaining are:
1. The presence of a fuel.
2. An appropriate oxidant in suitable proportion to the fuel.
3. Sufficient and suitable energy supplied (usually as heat) for the ignition of the fire.
4. The ability of the fuel and oxidant to react in a chain reaction that is self-sustaining.
• Once a self-sustaining fire has started, the heat that it generates is more than enough for this
purpose, thereby allowing continuous reignition to occur.
• Furthermore, removal of one or more of the four conditions above will put out an established
fire. This is the basis of all fire-fighting techniques.
• Distinction can be made between fires that have flames (i.e. plumes of burning gas) and fires
that smoulder (i.e. produce heat and light without the presence of flames).
(b) Flaming versus Smouldering
• In conventional fires, flaming may result from the flammable gas arising from pyrolysis (i.e.
the chemical breakdown under the influence of heat) of a solid fuel, such as wood or coal.
• Pyrolysis is a thermochemical decomposition of organic material at elevated temperatures in
the absence of oxygen (or any halogen). This reaction involves molecular breakdown of larger
molecules into smaller molecules in presence of heat.
• Pyrolysis is also known as thermal cracking, cracking, thermolysis, depolymerization, etc.
At any given temperature the molecule is in vibrating stage.
• Alternatively, it may be present as the result of the vaporisation of a liquid fuel, such as petrol,
or the fuel itself may be a gas, such as methane (the dominant component of natural gas).
• Smouldering usually takes place when solid fuels burn and takes place at the interface
between the solid fuel and the air. Smouldering means burning slowly with smoke but no
flame.
• Smouldering occurs if the ventilation and/or the heat supply is insufficient to support flaming
combustion. The flaming fire spreads at a much faster rate than the preceding smouldering
combustion.
• Many organic solid fuels (i.e. solid fuels based on carbon compounds), including wood,
pyrolyse in conventional fires to produce not only flammable gases but also a char.
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• Char is an impure carbon that remains after the gaseous pyrolysis products have ceased to
form and undergoes smouldering combustion.
• Well-ventilated char that is undergoing smouldering combustion may be very hot and produces
small flames that are mainly due to the gaseous combustion of carbon monoxide (CO).
• Completely pyrolysed smouldering char will not support the full flaming combustion
frequently observed during the char-producing pyrolysis process, unless fresh fuel is supplied
to the fire.
• If such a fire is allowed to spread, it may become sufficiently hot and ventilated to allow
flaming combustion to ensue (i.e. the fire may burst into flames).
(c) The behaviour of fire
1) Fires in rooms and similar compartments
• An unhindered burning fire taking place in a room that has a typical fuel load proceeds in a
fairly predictable fashion.
• However the time taken from the start of a room fire to its end varies significantly from one
incident to another.
• The sequence of events that occurs in a room fire is divided into a number of stages namely:
1. Ignition.
2. Growth.
3. Flashover
4. Post-flashover steady-state burning (also referred to as a fully developed fire).
5. Decay.
• The last of these stages ends when the fire stops burning as shown in Figure 8.1.
• Once ignition has occurred, the heat release rate of a given room fire (which is the power
transmitted out of the fire as heat) typically follows a pattern of increase, plateau and decline.
• These three phases correspond to growth culminating in flashover, post-flashover steady-state
burning and decay respectively.
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Figure 10.1: A typical graph showing the total heat release rate of a normal fire in a
furnished room plotted as a function of time. Note that a similarly shaped graph is
produced when the average temperature of the layer of hot gas within the room is plotted
as a function of time
[1] Ignition
• Ignition occurs when the four conditions required for a fire to start occur simultaneously in the
same place.
• The newly ignited fire may produce flames from the start or smoulder at the outset, bursting
into flames only once the heat release rate and ventilation are adequate for it to do so.
• Indeed, some smouldering fires never reach these conditions and so do not burst into flames,
and do not proceed through the sequence of events described here but smoulder until they self-
extinguish.
• In the vast majority of conventional fires, whether flaming or smouldering, ignition happens
when heat supplies energy at a sufficient rate to a suitable fuel that is already in contact with
the oxygen in the air.
• The heat that ignites the fire may be liberated by a number of processes. The most notable of
these are listed below:
1. Friction: e.g., when the head of a match stick is rubbed against a suitably rough surface or
when a mechanical bearing overheats due to insufficient lubrication.
2. Exothermic chemical reactions: e.g., during the self-heating of fuels (as happens in cases
of spontaneous combustion and fires caused by pyrophoric carbon), and, much more
commonly, in cases where an established fire causes another portion of fuel to ignite. Such
an established fire may be small, as in the case of a burning match, or large, for example a
raging house fire.
3. Electrical heating: e.g., when an electric current passes through a resistor. All normal
materials through which electricity passes offer some resistance and so will produce heat.
Conditions under which electrical heating can cause ignition temperatures to be reached
may happen when electricity passes through:
o a gas (in which case the current flow is referred to as an arc);
o a poor electrical contact in a wiring system;
o a heating element (e.g. as found in an electric cooker);
o the incandescent wire in a conventional light bulb;
o a conductor (such as an electrical wire) at a current that is in excess of that which it is
designed to withstand;
o A failing organic insulating material.
4. The nuclear reactions that occur in the Sun. These lead to the production of heat
radiation, which moves out into space in all directions from the Sun. This radiation delivers
an insolation rate (i.e. the rate at which solar radiation delivers energy per unit horizontal
area of the Earth’s surface) with a maximum of about 1 kW m–2. This is insufficient to
ignite frequently encountered fuels. However, the action of a reflective concave surface or
a transparent object capable of acting as a converging (i.e. convex) lens may focus the
Sun’s rays. This may produce a maximum energy delivery rate of 10–20 kW m–2, which is
capable of heating cellulose-based fuels/paper (e.g. newspaper) to their ignition
temperatures.
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• With the exception of fires caused by the self-heating of fuels, before ignition can occur energy
must be transmitted to the fuel that is to be involved in the fire. This energy is usually supplied
by heat, in which case, mechanisms by which it may be transmitted are thermal conduction,
convection and heat radiation.
• In some instances, one of these mechanisms dominates, while in others two or all three of them
play a significant role. For example, the ignition of a property that is adjacent to a house that
is on fire may occur principally because of heat radiation from the fire.
• In contrast, when the flame of a cigarette lighter is held under a piece of paper to set it on fire,
the heat transfer will occur by all three mechanisms.
• In many cases, a fire is ignited when a source of heat is physically moved into a position where
it can supply energy at a high enough rate to cause the ignition temperature of a fuel to be
reached.
• This physical movement may be deliberate, such as when an arsonist throws a lighted match
onto a petrol-soaked carpet.
• However, this is not always the case. It may occur, for example, when convection currents
carry a smouldering spark from an established fire into the air and it is then transported by the
wind to a new batch of fuel.
[2] Growth
• During the growth stage, fire development is not usually limited by the air supply but by the
rate at which new fuel items become involved.
• Consequently, as the progress of the fire is limited by the availability of fuel, the fire is referred
to as being fuel-controlled.
• During the early stages of a fire in a room, the combustion is normally limited to the fuel item
that was originally ignited.
• This will burn freely, producing a plume of hot gas that is carried upwards by convection until
it meets the ceiling, whereupon it will spread outwards until the walls are reached.
• At this point, the hot gas, which is depleted of oxygen and laden with the products of pyrolysis
and combustion, forms a layer at the top of the room that will become thicker with time.
Beneath this layer of hot gas, the air remains relatively fresh and cool.
• Typically, during the development of this layer, the fire will spread. The exact pattern that this
spread takes varies from fire to fire and is dependent on the position of items of fuel relative
to the fuel item that initially caught fire.
• If there is fuel above this item, it will typically ignite, allowing the fire to spread upwards and
outwards, producing a characteristic V-shaped char pattern.
• Heat radiation from the combustion of the first fuel item to burn will often elevate neighbouring
fuel items to their ignition temperatures, allowing the fire to spread horizontally.
• The flames in the plume of hot gas that rises from burning fuel items vary in length. If a given
fuel item, for example an armchair, were to burn on the floor of a room, its position in that
room would influence the length of the flames that it produces.
• Flames produced by the item burning against a wall would typically be longer than if the item
were burning in the centre of the room.
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• If the item were to burn in a corner, its flames would normally be longer still. The explanation
for this phenomenon lies in the rate at which air can be entrained in the rising plume of hot gas
within which the flames occur.
• A fire that occurs in an item resting on the floor in the middle of the room can draw in air from
all sides. This air supplies oxygen that allows the gaseous pyrolysis products to burn shortly
after their formation and cools the plume of gas. Both of these effects will tend to keep the
flames short.
• The plume of hot gas rising from the same fire occurring near to a wall is more restricted in
the directions from which it can entrain air.
• Consequently, the rate of air entrainment is comparatively low, meaning that the hot gases are
not cooled to the same degree and the pyrolysis products take longer to burn, resulting in flame
elongation.
• For the same reasons, the fire burning in the corner will have flames that are longer still. As
the fire grows, the heat release rate due to combustion will normally exceed the rate at which
heat is transferred out of the room.
• As a consequence, the temperature of the room will rise. However, throughout the growth
stage, the temperature differential between the hot gases in the upper part of the room and the
relatively cool, relatively normal air in the lower part will be maintained.
• Often the temperature in the layer of hot gas in the upper part of the room will become high
enough to allow the pyrolysis products and partially combusted material that it contains to
ignite.
• This ignition may happen due to flames, which may have become elongated because of their
proximity to a wall or corner, reaching the hot gas layer from below. Once the hot gas layer
ignites, the flame within it spreads at a very high rate (up to 3 to 5 m s–1).
• Consequently, within a period of a few seconds, all the room just beneath the ceiling may
become engulfed in flame and the temperature of the hot gas zone will increase appreciably.
• The spread of flames through such flammable products of combustion, during the progress of
a fire, is known as rollover or flameover. It may also be observed whenever these products
escape from a zone of oxygen depletion into more normal air, so long as they are hot enough
to ignite in their new environment.
[3] Flashover
• As the temperature of the hot gases in the upper part of the room increases, so does the rate at
which these transmit heat in the form of heat radiation.
• In a room of normal dimensions, irrespective of whether or not flameover occurs, if this gas
reaches a temperature of about 600°C, the radiant heat that it produces reaches floor level at a
rate of approximately 20kWm–2.
• This is sufficient to ignite cellulose-based fuels, such as wood and cotton. Consequently, at
this point, all fuel items in the room will burst into flames within a very short period of time.
• This phenomenon, in which all combustible items become involved in the fire, is known as
radiation-induced flashover, or just flashover.
• The high level of turbulent mixing that accompanies this generalized burning disrupts the
layered structure of the gases in the room that developed during the growth stage of the fire.
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• Flashover does not happen in all room fires. In order for it to occur, it is necessary for a layer
of hot gases to accumulate in the upper part of the room and for these gases to reach a
sufficiently high temperature. These conditions may not be met for a number of reasons.
Flashover will not occur if:
1. The room contains insufficient fuel;
2. The fuel present releases heat at an insufficient rate;
3. The level of ventilation is too low (insufficient ventilation will slow both the combustion
process and the heat release rate – in extreme cases, poor ventilation may even extinguish
the fire);
4. The flow of heat and/or gases out of the room is too great.
[4] Post-flashover steady-state burning (also referred to as a fully developed fire)
• Once flashover has occurred, all of the fuel present in the room is involved in the fire and its
heat release rate is maximal.
• At this stage, the fuel in the room will continue to burn at a rate that is determined by the
amount of air available (i.e. it is ventilation-controlled) until most of this fuel has been used
up.
• During this phase, because of the limited supply of oxygen, more gaseous fuel will normally
be produced by pyrolysis and partial combustion than can be consumed within the room.
Consequently, rollover flames are likely to occur as the hot smoke leaves the room.
[5] Decay
• As the available fuel is consumed, a point will be reached where the rate of air supply outstrips
the fuel supply rate and the fire again becomes fuel-controlled.
• Consequently, during this phase, as the fuel supply drops, so does the heat release rate. At the
same time, the amount of flaming combustion present will decrease as the ability of the
remaining fuel to form flammable pyrolysis products diminishes.
• Eventually, the fire will be dominated by smouldering combustion and, ultimately, the fire will
self-extinguish.
• In a room fire, decay can also occur under ventilation control if the air supply is so low that
the flames die down.
• Under these conditions, the concentration of flammable pyrolysis products may build up
sufficiently to create the conditions necessary for flashback to ensue if air is suddenly admitted
to the room.
2) Outdoor fires
• Outdoor fires ignite for the same reason and in essentially the same way as do fires within
rooms and similar compartments. However, once lit, the behaviour of outdoor fires is much
simpler than that of fires that occur in rooms.
• In a fire that is burning without the constraint of physical barriers:
1. Convection moves heat upwards.
2. Conduction moves heat away from its source via the material through which it is travelling.
3. Radiation allows heat to travel in all directions from its point of origin, unless it is absorbed
or reflected.
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• Importantly, under most circumstances, convection is the most efficient of these three
mechanisms at transferring heat from burning fuel to fuel that is yet to ignite.
• Consequently, fire will tend to move upwards through a three-dimensional matrix of solid fuel
much more rapidly than outwards or downwards. This will tend to produce a conical pattern
of fire development, which is narrowest at the bottom and widest at the top and which has the
seat of the fire at or very near to its base.
• Any vertical, or near-vertical, flat surface (e.g. a wall) that comes into contact with this will
tend to have a burn pattern on it that is approximately V-shaped. In objects that are partially
burnt through, these V-shaped patterns may also be evident, either in full or in part.
• Consider a small flaming fire that is lit in an open space, on flat, horizontal ground, on a still
day.
• The high temperatures of the fire will cause convection currents to be established that involve
a plume of hot gases rising above the fire and a flow of air from surrounding areas into the
base of the fire. The fire will move outwards in the direction of available fuel.
• This process will occur fairly slowly because the convection-driven stream of air that is moving
towards the base of the fire almost exclusively transports heat away from the direction of fire
propagation, leaving conduction and radiation as the only means of raising surrounding fuel
items to their ignition temperatures.
• Under these circumstances, if the fire is evenly surrounded by sufficient fuel with identical
burning characteristics, a circular fire spread pattern will result.
• If this same fire were to occur on a slope, however, the convection currents would cause hot
gases to heat any fuel on its up-hill side, thereby facilitating the much more rapid spread of fire
in this direction.
• This will typically produce a fan-shaped fire spread pattern. Note that slow down-hill burning
will also occur due to the mechanisms described in the previous paragraph.
• In addition, any burning items, such as the branches of trees, that fall down-slope out of the
burning zone, may well create new centres of ignition, each of which will typically produce its
own fan-shaped burn pattern.
• Ambient wind has the effect of pushing the plume of hot gases that arise from the top of a fire
towards the ground. This, like the effect of a slope, will help the spread of fire, in this case in
a downwind direction.
• On flat, horizontal land that is evenly covered in fuel of similar burning characteristics, wind
that is blowing strongly in one direction will typically produce a burn pattern that is
significantly longer than it is wide. It is noteworthy that wind-driven sparks from one fire can
kindle a new fire in a downwind location.
• Fierce fires produce significant amounts of radiant heat. This can cause fire to move from one
place to another, even though the intervening space contains no fuel. This can result in one
burning building setting another on fire.
(d) Fire scene investigation
• A fire investigation will have a number of aims. Typically these include:
1. The identification of where the fire started (known as the seat of fire or point of origin).
2. The determination of the cause of the fire and whether it was intentionally started.
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3. The establishment of legal liabilities associated with the fire.

4. The determination of the factors that contributed to and controlled both the fire’s spread
and the production of heat, flame and smoke during its progress.
5. The identification of any health and safety issues that arose during the fighting of the fire.
6. The identification of dangerous practices, materials and manufactured items that
contributed to the start or progress of the fire.
7. The collection of data for use by policy makers.
• In addition, in cases in which claims are to be made against insurance policies, loss adjusters
will be instructed by the insurance companies involved to assess the extent and monetary value
of fire damage.
• Fire scene investigations will achieve their optimum effectiveness only if they are carried out
with due care, diligence and expertise, and in an ethically and legally acceptable fashion.
• A fire will start whenever a suitable fuel and oxidant, which are capable of a self-sustaining
chain reaction, are brought together in appropriate proportions and provided with sufficient
energy for ignition to occur.
• A statement of how these conditions arose at the start of a particular fire is a statement of the
cause of fire.
• Each fire scene that is investigated should be treated as if it were a crime scene until unless it
is established that this is not the case.
• This precautionary approach is wise for the following reason. If an investigation establishes
that it is likely that arson has occurred, and the processing of the site has not been carried out
as if it were a crime scene, then the value to any subsequent prosecution case of evidence
obtained from the scene might be severely limited.
• From a forensic science perspective, it is of paramount importance to investigate a fire scene
with a view to establishing whether the fire was caused intentionally.
• In order to do this, it is normally necessary to determine both the seat and the cause of the fire.
(e) Establishing the cause of a fire
• Most fires start when a source of ignition supplies sufficient energy, usually as heat, to light
a fuel that is already in contact with a suitable oxidizing agent (in the vast majority of cases,
this oxidizing agent is oxygen (O2), provided by the air).
• The key to establishing the cause of the fire is normally the finding of its seat and subsequently
the identity and/or location of the fuel item that started the fire.
• In some instances, direct evidence of such fuel item (source of fire) may be present or absent
as when an arsonist starts a fire with a cigarette lighter that he or she subsequently takes away
when leaving the scene.
• From a forensic science point of view, the most important feature to be established concerning
the cause of a fire is whether the fire was deliberately started, as the crime of arson necessarily
involves this act.
• There are many indications of deliberate fire setting, some of which are essentially unequivocal
(e.g. finding the remains of an incendiary (flammable) device at the seat of the fire) while
others are circumstantial. Some of these indicators may be found at the scene and others
elsewhere.
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• Clearly, no two instances of deliberately set fires are the same and the exact nature and number
of indicators left by the fire setter will vary from case to case.
• However, there are a number of more commonly encountered classes of indications of the
deliberate starting of fire. Notably these include the following:
1. Lack of evidence for a natural or accidental cause of the fire (note that this in itself is not
normally sufficient to establish that the fire was deliberately started; also, attempts may be
made to make deliberate fires appear to be accidental by, for instance, starting them with,
or near to, heating appliances, cookers, machinery, etc.).
2. Evidence that a flammable liquid was among the first fuels to be ignited. Such evidence
may include:
o burning at low elevations within the fire-damaged area;
o burn patterns that are localized (restricted);
o burn patterns that show sharp, curved edges;
o the presumptive detection of vapours of hydrocarbon (or other) liquid fuels from locations
(e.g. cracks between floorboards) and materials (e.g. carpet underlay) that may have
offered the liquid protection from being burnt (such presumptive detection may be
achieved by using the human sense of smell, trained ‘sniffer’ dogs (known as hydrocarbon
search canines) and/or portable electronic detectors);
o detection and identification of liquid fuel by the laboratory analysis of samples retrieved
from the scene (this is the only definitive evidence for the presence of flammable liquids).
Note that other materials (such as burning plastics and dropdowns) can mimic the burn
patterns produced by flammable liquids; also the presence of flammable liquid does not
prove deliberate fire setting as such liquids may have been innocently stored at the scene
prior to the fire.
3. Evidence of the presence of an unusually high quantity and/or quality of fuel at the seat of
the fire (which may be exhibited, for example, in unexpectedly severe destruction).
4. Objects suspiciously out of place, for example:
o piles of fuel items (which may be evidenced by the presence of otherwise inexplicably high
numbers of hinges, drawer locks, furniture springs, etc. within the ash);
o containers that may have held flammable liquids found in unexpected places;
o tools or other equipment that might have been used to break into a property or to perpetrate
sabotage;
o filing cabinet or desk drawers left open (to aid the combustion of documents);
o the substitution of good furniture for poor-quality items; the prior removal of objects of
sentimental or monetary value (e.g. photographs, jewellery or share certificates);
o otherwise inexplicable escape of pets;
o doors, windows or roof space access hatches wedged or left open;
o electrical switches left on that would normally be expected to be off;
o heating system thermostats set unusually high (to improve ventilation);
o objects placed so as to hinder the activities of the fire brigade;
o Transparent or reflective objects that are capable of focusing the Sun’s rays left in a
position where they could lead to the ignition of fuel.
5. Unusual damage, for example:
o partition walls/ceilings/floors broken through to improve ventilation and aid the spread of
the fire;
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o signs of break-in;
o disabled fire and/or intruder alarms;
o Indications of sabotage (e.g. cut fuel pipes, electrical supply systems that have been
tampered with).
6. Evidence that a crime was committed at the scene prior to the fire.
7. A history of fires in the building or neighbourhood concerned and/or a similar history
associated with anyone who stands to gain from the fire.
8. A familiar face in the crowd watching the fire or someone leaving the scene (most people
are attracted by the spectacle of a fire and will want to observe it).
9. The presence of one or more incendiary devices or their remains. Examples of such devices
include:
o incendiary devices with a time-delayed action (which may be very simple, such as a
balloon, or similar container, filled with flammable liquid and left above an ignition
source);
o Molotov cocktail (i.e. a container filled with a flammable liquid and equipped with an
ignition device designed to light the liquid when the container is broken – Molotov
cocktails are usually thrown);
o A purely chemical means of causing ignition.
10. Fire safety systems such as automatic fire detection systems and/or sprinkler systems
turned off or made inoperative.
11. Multiple fire seats (if this occurs, intact but failed incendiary devices may be found
elsewhere on the premises).
12. Evidence of lines of flammable material (known as trailers) deliberately placed so as to
spread the fire from one location to another.
13. Mismatches between statements made by, for example, the owner of the fire-damaged
property (including any contents inventory provided) and the sequence of events known to
have taken place during the incident and/or the physical evidence left after the fire.
14. Indications of a motive for arson among any people who had the potential to start the fire.
15. Patterns of destruction in any one fire or patterns that emerge when the characteristics of
several fires are compared that suggest that an arsonist may be at work.
• In cases of arson, these indicators not only help establish the cause of the fire, but may also
give an insight into the possible motives of the arsonist. For further information on this
important topic.
• Laboratory-based chemical analysis of samples connected with cases of suspected illegal fire
setting (whether successful or attempted) can provide valuable evidence.
• Such samples may be those recovered from the scene of a fire (whether actual or apparently
intended), any suspects involved in the case and/or the possessions of those suspects.
• The initial purpose of such analysis is to establish whether the sample contains a substance that
may have been used with the aim of increasing the rate of fire spread (i.e. a potential fire
accelerant). Furthermore, laboratory analysis may:
1. Identify the chemical nature of any fire accelerant detected;
2. Allow the amount of any such material present in the sample to be established; and/or
3. Provide points of comparison between samples taken from different locations.
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• The last of these may be of particular importance in helping to establish a link between a
suspect and an apparent crime scene.
• In cases of arson, the fire accelerants most commonly detected by laboratory analysis are liquid
fuels. Among these, liquid petroleum distillates are the most frequently encountered, although
other flammable liquids, such as short-chain alcohols (e.g. ethanol), may also be used as
accelerants.
• The most prevalent petroleum distillate found in this context is petrol but others, such as
paraffin or diesel oil, may be employed by an arsonist. Each of these distillates is a complex
mixture of volatile hydrocarbons (a hydrocarbon is a compound that contains only hydrogen
and carbon).
• Gas chromatography (GC) is the main technique used to analyze samples suspected of
containing liquid fire accelerants.
• This is because as a means of analyzing volatile compounds, GC is highly sensitive (and so
can be used for the analysis of trace as well as bulk samples).
• It is also capable of isolating, and potentially identifying, individual components of mixtures
of such compounds.
• Analytical data that show a match between an accelerant recovered from an arson scene and
flammable liquid found in possession of a suspect do not provide proof that the individual
concerned perpetrated the crime in question because the analysis of flammable liquids reveals
class characteristics and not individualizing ones
B. Analysis of Explosions and Explosives
(a) Explosions
• A conventional fire may be preceded or be accompanied by an explosion or explosions which
may occur if:
1. There’s a deliberate or accidental discharge of an explosive device.
2. A mixture of air and a flammable gas (e.g. natural gas), dust (e.g. flour or coal dust) or
vapour (e.g. petrol vapour) is ignited.
• For example, an arsonist who douses the inside of a property with petrol prior to setting it
alight may as well blow him- or herself up when striking the match. Flammable vapour
explosions are highly destructive.
• An explosion or explosions may accompany a conventional fire if, during the course of a fire,
a container of flammable liquid or gas is heated.
• This will cause the pressure in the container to rise and, if it bursts, a cloud of flammable gas
or rapidly vaporizing flammable liquid may be discharged into the vicinity of the fire.
• An explosion will then occur if the gas or vapour mixes with sufficient oxygen from the air
and is then set alight, most commonly by the fire.
• A smouldering combustion can cause an explosion if solid fuel that is capable of sustaining
flaming combustion is held within a compartment, such as a room, that is poorly ventilated.
• Under these circumstances, the fuel will pyrolyse and the concentration of flammable gaseous
pyrolysis products will rise within the compartment.
• If the compartment is then breached by, for example, a fire-fighter breaking a window, the
sudden access of fresh air may fulfill the remaining condition for intense flaming combustion,
namely the presence of an oxidant in sufficient amounts.
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• If this occurs, an explosion can result that is variously known as a smoke explosion, flashback,
backdraft or ventilation-induced flashover.
• Regrettably, the destructive nature of explosions has been used by a number of terrorist
organizations and criminals as a means of furthering their particular aims.
• Explosions have been used to terrorize communities, murder individuals, damage property and
facilitate theft (as in the case of safe-breaking).
(b) What are explosions?
• An explosion is a manifestation of a sudden release of energy. It may be defined as the violent
effect produced by the rapid build-up of gas pressure at a location because of the sudden
liberation of energy and, in most cases, gas at that location.
• The process that leads to a given explosion may be chemical, mechanical, thermal, electrical
or nuclear in nature. In the forensic context, explosions directly caused by chemical reactions
are the most interesting.
• Mechanical explosions occur when containers of pressurized gas and/or liquid burst to liberate
gas. Such explosions may happen, for example, in a fire or when a system that is designed to
be pressurized from within becomes over-pressurized (e.g. a vehicle tyre that is over-inflated).
• Thermal explosions may be produced when a liquid is rapidly brought to its boiling point by
contact with another liquid that is significantly hotter than it, such as may happen if water and
molten steel are allowed to meet.
• An electrical explosion results if the heating effect of the discharge of electricity through an
electrically insulating material is sufficiently intense. Nuclear explosions are beyond the scope
of this topic and will not be discussed here.
• Explosions due to chemical reactions can be subdivided into two types, namely deflagrations
and detonations.
• During a deflagration, the speed at which the reaction front moves through the explosive is less
than the speed of sound in that material.
• In a detonation, the speed at which the reaction front moves through the explosive is greater
than the speed of sound in that material.
(c) What are explosives?
• Any substance that is capable of producing an explosion is known as an explosive. In our
discussion, the term explosive is used to denote any one of those materials that can produce
explosions as a consequence of chemical reactions.
• Clearly, any substance of this type must be able to undergo a chemical process that rapidly
releases energy and, ideally, gas. To trigger an explosion, this process must be initiated.
• Initiation requires the supply of sufficient energy, in a utilizable form, to at least some part of
the explosive.
• However some explosive materials may liberate sufficient energy by internal chemical
reactions to cause an explosion to be initiated without the input of energy from outside.
• That is, they spontaneously explode (e.g. mixtures containing Sulphur and a chlorate are
notorious for doing this). Clearly, this is not a desirable characteristic in useful explosives.
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• In order to be an explosive, a material must be capable of propagating the explosive chemical

process through itself from the site of initiation.
• There are many explosives known that consist of suitable oxidizing and reducing agents that
are present in intimate combination and in appropriate proportions.
• This combination may be achieved by the physical mixing of materials that have oxidizing
properties with those that have reducing ones. Typical oxidizing materials used for this purpose
include inorganic nitrates, perchlorates and chlorates.
• Suitable reducing materials, which may be thought of as fuels, include Carbon, Sulphur,
hydrocarbons, carbohydrates (e.g. sugar) and finely divided metals (e.g. Aluminium).
• Gunpowder (also called black powder) is the archetypal explosive of this sort. It is a mixture
of saltpetre (potassium nitrate), charcoal (carbon) and Sulphur.
• Another commonly encountered explosive mixture is ammonium nitrate and fuel oil
(ANFO), which is used as a blasting agent in quarries.
• An alternative means of achieving the intimate combination of oxidizing and reducing agents
is to synthesize a compound, parts of which are oxidizing in nature while others are reducing.
• Such compounds tend to produce a more dramatic explosive effect than do physical mixtures
of oxidizing and reducing materials. They include:
1. 2,4,6-trinitrotoluene (TNT);
2. Cyclotrimethylenetrinitramine (RDX);
3. Glyceryl trinitrate (Nitroglycerine);
4. 2,4,6-trinitrophenylmethylnitramine (tetryl);
5. Cyclotetramethylenetetranitramine (HMX); and
6. Pentaerythritol tetranitrate (PETN).
• It is important to realize, however, that many explosive formulations that contain explosive
compounds are, in fact, blended products. These blends are made in order to modify the
characteristics of the pure materials from which they are formulated.
• For example, nitroglycerine is rendered much safer to handle by combining it with an inert
absorbent such as kieselguhr (a diatomaceous earth – a geological material produced by the
sedimentary deposition of diatoms’ skeletons) to form dynamite (known as straight dynamite
in the United States).
(d) The classification of explosives
• High explosives normally detonate and produce a shattering effect. They are used in both
military and industrial applications where blast is required.
• High explosives do not normally need to be confined in a container in order to explode although
a number of military munitions are normally filled with this type of explosives, including
shells, mines, bombs and grenades.
• When they explode, such munitions produce both blast and rapidly moving fragments of their
casings. PETN- and RDX-based formulations are commonly used in military blasting
applications.
• Frequently encountered industrial blasting formulations may be based on ammonium nitrate
and/or nitroglycerine.
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• These include ANFO and dynamite and blends based on mixtures of either nitroglycerine and
nitrocellulose, or nitroglycerine, nitrocellulose and ammonium nitrate. High explosives are
also called detonating explosives.
• Deflagrating explosives (formerly known also as low explosives) are those that will not
normally detonate.
• In order to explode – rather than burn – they need to be confined or contained. When they do
explode, their action is better described as pushing rather than shattering but their action can
be devastating.
• In many applications, a distinction can be drawn between primary and secondary explosives.
In such applications, the former of these materials is significantly easier to initiate than the
latter.
• The devices used in these applications contain a small amount of primary explosive and a main
charge consisting of a larger quantity of secondary explosive.
• During use, initiation of the primary explosive liberates a sudden burst of energy that is
sufficient to initiate a reaction in the main charge.
• This arrangement is used in firearms cartridges in which a percussion-initiated primary
explosive (the primer) is used to set off the main charge (the propellant).
• It is also used in devices in which a main charge of high explosive (secondary explosive) is
initiated with a detonator, which contains primary explosive.
• It is also useful to recognize a distinction between condensed explosives and dispersed
explosives.
(e) Explosion scene investigation
• The investigation of the scene of an explosion has, in most cases, the potential to become the
investigation of the scene of a serious crime. Consequently, until and unless there is a reason
to treat it otherwise, it is wise to process the scene of an explosion as if it were that of a serious
crime.
• Explosion scene investigations differ from those of other scenes in a number of important
respects. This is because of:
1. The unusual risks to the investigators, the emergency services and the public that are
frequently posed by explosion scenes (although it should be noted that fires and other large
scenes often also pose significant risks);
2. The immense destruction that is often wrought by an explosion and any subsequent fire;
3. The need to establish the cause of the explosion (i.e. what exploded and why it did so).
• The principles, methods and management structures used to examine an explosion scene are,
in essence, the same as those used to process any other major crime scene.
• Irrespective of the likely cause of the explosion, the preservation of life and the maintenance
of safety are of paramount importance. For scenes that may involve explosive device(s), the
site is not entered for the purposes of examination until it is declared safe by explosives expert
personnel.
• The investigation of an explosion will normally allow a number of conclusions to be drawn,
although the degree of certainty associated with these may vary from conclusion to conclusion
and from incident to incident.
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• Importantly, these conclusions will normally include an assessment of whether the explosion
occurred as the result of an accident or a deliberate act and the type of explosion involved (i.e.
chemical, mechanical, thermal, electrical or nuclear).
• In instances of explosions caused by chemical reactions, the investigation will normally reveal
whether the explosive was condensed or dispersed and whether it deflagrated or detonated.
• Note that the process of elimination is an important tool in the investigation of explosions as it
can reveal truths that are not directly observable.
• For example, if all natural and accidental causes of an explosion can be eliminated, then it
must be concluded that the explosion occurred as the result of a deliberate act.
(f) The analysis of explosives
• Laboratory-based chemical analysis of samples connected with cases of suspected illegal use
of explosives can provide valuable evidence.
• Such samples may be those recovered from the scene of an explosion (whether actual or
apparently intended), any suspects involved in the case and/or their possessions.
• The purpose of such analysis is:
1. Initially to establish whether the sample contains an explosive.
2. To identify the chemical nature of any explosive detected.
3. To establish the amount of explosive present in the sample and/or provide points of
comparison between samples taken from different locations.
4. To help establish a link between a suspect and an apparent crime scene or between a
number of seemingly independent incidents.
• During chemical analysis, a distinction must be made between trace and bulk quantities of
questioned material. Strictly, bulk quantities are those that may be weighed while trace
quantities are those that are too small to be seen with the naked eye.
• Examples of trace quantities include the explosive residues that may be recovered from a
suspect’s hands after he or she has handled explosives.
• This distinction between trace and bulk amounts of questioned material is important because
bulk samples have the potential for contaminating items containing trace quantities of
incriminating material.
• Therefore, it is good practice to completely separate those facilities that handle and analyse
bulk explosives from those that deal with trace samples.
• In the case of explosives, another reason for this distinction is that great safety precautions
need to be taken during the analysis of substantial bulk samples because of both the explosion
hazard that they pose and, in many cases, their toxic nature.
• A wide range of techniques may be used for the analysis of samples that are suspected of being
composed of, or contaminated with, explosives. These include:
1. The physical testing of the explosive properties of the sample;
2. Spot tests (i.e. chemical tests devised to produce visible changes in the presence of small
amounts of specific types of chemical and that may indicate the presence of an explosive);
3. Light and scanning electron microscopy (the latter of which can be coupled with energy
dispersive X-ray analysis (EDX), allowing the determination of the elemental composition
of the sample);
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4. Infrared spectroscopy – a technique that can allow the detection of the presence of
individual chemical species or chemical functional groups, and that provides multiple
points of comparison between samples;
5. Various forms of chromatography, including:
o Thin-Layer Chromatography (TLC);
o Gas Chromatography (GC) (which may be linked to mass spectrometry (MS) to give
GC–MS);
o High-Performance Liquid Chromatography (HPLC); and
o Ion Chromatography (IC);
6. Atomic absorption spectroscopy (AAS) – a technique that can be used to quantify most
elements that may be present in a sample.
• The methods that are selected for a given application will depend on a number of factors such
as:
1. Whether the sample is believed to contain bulk or trace amounts of explosive (e.g. the
physical testing of the sample’s explosive properties can be carried out only on bulk
samples);
2. The nature of the explosive material that is suspected to be present (e.g. if it is believed
that the material might contain an ionic oxidising agent, such as nitrate (NO3–), ion
chromatography might be employed, whereas if it were to contain a relatively volatile
organic explosive compound (e.g. nitroglycerine) gas chromatography might be chosen);
3. The sample matrix within which the explosive is collected (e.g. if the matrix is soil dug
from the crater left by a bomb, it is likely that some form of sample pre-treatment –
designed to largely separate any explosive present from its original matrix – will be used
prior to the application of the main analytical technique(s)).
• A range of methods based on GC and HPLC has been devised for the analysis of explosive
compounds such as nitroglycerine or PETN.
• Given the superb sensitivity that may be achieved by these techniques, they are the methods
of choice for the analysis of such compounds at trace levels.
• When carrying out the laboratory analysis of samples that are suspected of containing
explosives, the parallel analysis of appropriate control samples may be of utmost importance.
• In this context, the control samples chosen will be ones intended to either:
1. Replicate the matrix within which the explosive is believed to reside in the questioned
samples; and/or
2. Check that the environment from which the questioned samples were taken, or in which
they were stored or handled, could not produce a false-positive result. For example,
consider a case in which swabs were used to take samples from the hands of someone
suspected of handling explosives.
• If these were analyzed and shown to contain traces of explosive and the case were to go to
court, defense lawyers could argue that the traces of explosive could have arisen because of
contamination from:
1. The swabs themselves;
2. Any solvents or reagents used to damp the swabs prior to their contact with the hands of
the suspect or during the subsequent processing of the swabs; and/or
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3. Any surfaces (laboratory bench tops, the gloves worn by the analyst, etc.) with which the
swabs could have been brought into contact during their processing.
• However, the defense’s argument could be shown to be invalid if the laboratory had also:
1. Analysed the following control samples:
o swabs that were identical in all respects (including being damped with any solvent or
reagent employed) to those used to take samples from the suspect except that they had
not been in contact with the suspect’s hands; and
o swabs taken from all surfaces from which contamination could have occurred;
2. Carried out this analysis of control samples, using techniques, solvents, reagents and
equipment identical to those used for the questioned samples, and by this means established
that none of the control samples showed any traces of the explosive found in the questioned
samples.
• It is important to realize that analytical data that show the presence of explosive material are
not in themselves proof that a crime has been committed nor are they proof that a given
individual participated in a proven crime.
• Incriminating chemicals that may be part of an explosive formulation often have other
legitimate uses (e.g. ammonium nitrate is both a component of the explosive ANFO and used
as a plant fertiliser).
(g) Patterns of explosion damage

• Explosions due to explosives cause damage by displacing and permanently distorting objects
as well as by the heat that they release and the fires that may follow.
• At the scene of an explosion, the analysis of the patterns of damage can be highly informative.
A number of features of the damage are of value. Notably, these include those that provide
indications of:
1. The direction in which the blast and/or fragments travelled at any given point in the scene (an
example of the former type of feature is the bending of a pipe, while an example of the latter
is the penetration of a relatively soft material by a fragment of bomb casing, thereby leaving
an elongated hole – extrapolation from this would allow the direction of travel of the fragment
to be established);
2. The location of the region of maximum damage;
3. The maximum pressure that was exerted by the explosion at various points in the scene. The
construction of a plan of the scene on which direction indicators are marked will frequently
allow the investigator to establish the location of the place where the explosion started or so
called the explosion centre. This is possible because the explosion centre will be revealed as
the place from which blast and/or fragments of any explosive device originated. Locating the
explosion centre is of considerable value because:
(a) It will coincide with the place where the explosion was initiated, thereby assisting the
investigation by indicating where to look for evidence of the means by which initiation
occurred;
(b) In cases that involve condensed explosives, the vicinity of the explosion centre is a likely
place to find explosive residues in concentrations that may be detected by laboratory
analysis;
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(c) This is the origin of the trajectory taken by any fragments of any bomb involved (and/or
any container used to conceal any bomb) thereby providing information about the likely
location of such fragments;
(d) When used in conjunction with other observations based on patterns of damage, it will
allow experts to discern strong indications as to whether a given chemical explosion was
caused by a condensed explosive or a dispersed one, and whether this explosive detonated
or deflagrated.
• In the case of condensed explosives, one of the parameters that may be estimated from an
analysis of damage patterns is the weight of the charge.
• There are a number of ways in which this may be done. For example, for all but small charges,
crater dimensions can be used, thus:
𝑑3
𝑊≈
16
• Where W is the weight of the charge in kilograms and d is the diameter of the crater in metres.
(h) Evidential value of debris from explosive devices
• The debris from any explosive device may include portions of any detonator employed, the
wrappings from cartridges of commercial explosive (if used), and/or fragments of any
electrical or mechanical timing devices, remote control or victim-actuated mechanism present.
• The examination of such fragments may reveal a number of valuable facts. Importantly, it may
allow:
1. The materials from which the explosive device was made to be identified (e.g. the
laboratory analysis for trace levels of explosive not consumed in the incident can reveal
the nature of the explosives involved, as may the presence of trade marks on wrappings);
2. The construction of the device to be deduced;
3. The likely manner in which it was triggered to be established;
4. In some cases, the likely identity of one or more people involved in the incident (e.g. from
fingerprints found on one of the device’s component parts, such as sticky tape used to hold
it together).
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TOPIC 8: ANALYSIS OF FIREARMS
A. History of Gunpowder and Firearms

• Ballistics is the study of projectiles (bullets) and firearms. A firearm is a weapon, such as a
gun, capable of firing a projectile using a confined explosive. Ballistic evidence helps police
answer many questions pertaining to a crime scene. These questions include:
1. What type of firearm was used?
2. What was the caliber of the bullet?
3. How many bullets were fired?
4. Where was the shooter standing?
5. What was the angle of impact?
6. Has this firearm been used in a previous crime?
• More than one thousand years ago, the Chinese invented gunpowder. Gunpowder is potassium
nitrate (saltpeter), charcoal, and sulfur.
• When ignited, it expands to six times its original size, causing a violent explosion. The Chinese
used it to make fireworks and to shoot balls of flaming material at their enemies.
• Years later, in 14th-century Europe, inventors learned they could direct the explosive force of
gunpowder down a cylinder to move a deadly projectile, an object that is launched through the
air.
• The projectiles launched from these early firearms were very effective in piercing suits of
armor and wounding the enemy at a great distance.
• For a firearm to work reliably, it must effectively ignite the gunpowder. The earliest firearms,
matchlock weapons, had wicks to carry a flame to the gunpowder (Figure 9-1).
Figure 9-1: Matchlock weapons used wicks to ignite the gunpowder.
• Later, matchlock weapons were replaced by flintlock weapons, which used sparks from a chip
of flint instead of wicks to ignite the powder, allowing them to work even in damp weather.
• These weapons were muzzle-loaders, which meant that the user put the gunpowder and the
projectile down the firearm’s barrel (muzzle) and packed them into position.
• Percussion firing replaced the flintlock method with the introduction of the cartridge, a case
that holds a bullet (a pointed projectile), a small amount of primer powder, and the gunpowder
(Figure 9-2).
• A hammer hit the primer powder, which exploded, igniting the gunpowder. From the late
1500s, firearms that used this method were more effective than flintlocks in shooting the bullet
in the desired direction.
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• Cartridges were loaded into the gun from the opposite end of the barrel—the breech. These
breech-loading firearms could be loaded for firing more quickly than the older, muzzle-loading
firearms.
• Did you know? Much of the credit for the modern firearm is given to French army officer and
inventor Henri-Gustave Delvigne.
• In the 1820s and 1830s, he experimented with innovative rifles, bullets that expanded to fit
rifled barrels, and other advances in firearms.
B. Long Guns and Handguns

• Modern firearms are divided into two basic types—long guns and handguns. Long guns, such
as rifles and shotguns, require the use of two hands for accurate firing. Shotguns are often used
for hunting game birds and can fire many small pellets at once.
• Rifles fire bullets, whereas shotguns can fire either small round pellets (shot) or a single
projectile called a slug.
• Handguns fired with one hand are called pistols. American inventor Samuel Colt developed
and patented a model in 1835.
• Colt’s weapon, unlike its predecessors, had a cylinder that could be loaded with several
cartridges and fired in rapid succession. It was called a revolver, because the cylinder that held
the cartridges turned as it fired.
• Today, handguns can be further classified as revolvers or semiautomatic firearms. Revolvers
hold six cartridges in the cylinder. The semiautomatic permits the loading of up to 10 cartridges
into a magazine (clip), which is then locked into the grip of the firearm.
• Semiautomatic weapons, which fire only one bullet per pull of the trigger, differ from fully
automatic weapons, which fire repeatedly as long as the trigger is pressed. In both, the empty
cartridge ejects and the next cartridge advances automatically.
C. Firearms and Rifling

• An archer will hit a target with greater accuracy if there is a twist on the end of the arrow
feathers. This same principle of twisting the projectile is part of the design of “rifled guns” or
rifles.
• The word rifle originally referred to the grooves, or indentations, in the rifle’s barrel. The
ridges, or raised areas, that surrounded the grooves are called lands.
• Within the gun’s barrel, lands and grooves cause a bullet to spiral when exiting the barrel of
the gun, much in the same way a football spirals when thrown. This rifling pattern left on the
bullet is specific to the firearm.
• Today, technology allows us to identify the patterns of land and grooves in pistols and rifles
(Figure 9-1).
• Ballistics experts realize that, even as guns of the same model are produced, the tools used to
make the rifling within the gun’s barrel wear the metal in such a way that each gun has a unique
pattern.
• It is impossible to produce two identically rifled gun barrels. As a gun is fired, the barrel marks
each bullet with its own unique pattern.
• Therefore, a bullet can be matched to the specific gun from which it was fired.
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Figure 9-1: Bullets fired from a firearm show patterns of lands and grooves that match the
barrel pattern of the firearm.
• Did you Know? Samuel Colt’s revolver gained popularity so slowly that his first company
failed. He gained success with Mexican War orders for Colt .45s.
• His weapons soon became popular with civilians on the frontier and with the Texas Rangers.
D. Bullets, Cartridges, and Calibers
• A bullet is a projectile propelled from a firearm. Bullets are normally made of metal. The term
bullet is often incorrectly applied to the cartridge, which includes primer powder, gunpowder,
the bullet, and the casing material that holds them all together.
(a) Anatomy of a Cartridge
• The typical cartridge is composed of the following parts (Figure 9-2):
1. The bullet (the projectile) can be composed of lead, copper, or combinations of various
metals. It can be metal-jacketed, hollow-pointed, or even plastic-coated.
2. The primer powder mixture initiates the contained explosion that pushes the bullet down
the barrel. The primer is struck by the firing pin of the firearm. The pressure causes the
powder to ignite. The firearm’s firing pin may strike the bottom of the cartridge casing in
the centerfire cartridge, or it might strike anywhere on the rim of the rimfire cartridge.
3. The anvil and flash hole provide the mechanism of delivering the explosive charge from
the primer powder to the gunpowder.
4. The headstamp on the bottom of the cartridge casing identifies the caliber and manufacturer
(Figure 9-3).
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Figure 9-3: The typical center fire Figure 9-3: Ammunition has a headstamp—
cartridge. either a logo or name identifying the caliber
and manufacturer. The headstamp is located on
the bottom of the cartridge casing. Shown is
the
headstamp of a Luger 9mm.
• Did you know? “Magnum” or “special” associated with the cartridge refers to the use of a
higher energy gunpowder or more gunpowder.
• “Rim fire” or “center fire” refers to where on the rear of the cartridge the firing pin strikes
the cartridge casing—either along its rim or in the center.
• Smaller-caliber cartridges tend to be rim fire, while larger cartridges are usually center fire.
Generally, center-fire cartridges are more powerful than rim fire cartridges.
(b) How a Firearm Works
• The sequence of events in the firing of a bullet is (Figure 9-4):
1. Pull the trigger and the firing pin of the firearm hits the base of the cartridge, igniting the
primer powder mixture.
2. The tiny explosion—not much more than a spark—of the primer powder mixture on the
anvil delivers a spark through the flash hole to the main gunpowder supply.
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Figure 9-4: The sequence of events in the firing of a bullet.

3. The main gunpowder supply ignites, and the pressure of the explosion pushes the bullet
from the casing and into the barrel of the firearm. The amount of gunpowder and the mass
of the projectile in a cartridge determines the speed of the bullet.
4. The bullet follows the lands and grooves pattern of the barrel and begins its spiral before it
leaves the barrel.
• Did you know? Shotgun shells are measured in gauge. The 10-gauge, 12- gauge, 16-gauge, or
20-gauge refers to the number of round lead balls per pound of load in the shot.
• The larger the gauge number, the smaller the inside diameter of the barrel would be. A 12-
gauge shotgun will propel fewer (but larger-sized) lead shot than a 20-gauge shotgun.
(c) Caliber of the Cartridge
• Bullets (and their cartridges) are named by caliber and length. The caliber is a measure of the
diameter of the cartridge.
• Some common calibers include: 0.22, 0.25, 0.357, 0.38, 0.44, and 0.45. These cartridges are
usually measured in hundredths of an inch. Thus, a .45-caliber cartridge measures 45/100 of
an inch in diameter (almost ½ an inch).
• The 0.357 cartridge is 35.7/100, or 357/1,000 of an inch. The European method of naming
firearm caliber uses the metric system for measurement of cartridge diameter. Nine-millimeter
firearms fire 9 mm bullets.
• Caliber also refers to the diameter of the inside of a firearm’s barrel. Because the bullet moves
through the barrel, the caliber of ammunition should match the firearm that shoots it.
• If a bullet is removed from a wound or crime scene, its caliber can link it to the weapon used
to fire it.
(d) The Study Of Bullets And Cartridge Casings
• A significant part of ballistics involves examining used bullets and their spent cartridge casings
for telltale markings left on them by the firearm that shot them.
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• For example, as a gun is fired, the barrel marks each bullet with its own unique pattern of lands
and grooves. By examining the lands and grooves, a bullet investigators can match to the gun
from which it was fired (Figure 9-4).
• Investigators compare bullets and spent cartridge casings from a crime scene with bullets and
spent cartridges shot from the suspected firearm.
• To get a known bullet for comparison, investigators test-fire the weapon into a water tank or
gel block.
• This captures the bullet without damaging it. Then, they can compare the markings on known
bullets with those on the suspect bullets.
Figure 9-4: The barrel of a firearm has a unique pattern of rifling, which leaves a
matching pattern of lands and grooves on the bullets it fires.
• Did you know? According to a recently released study, (November 2007) evidence matching
the chemical composition of a crime scene bullet to that of a specific production lot has been
discredited.
• It apparently had no scientific credibility, although it has been used as evidence in criminal
proceedings for over forty years!
• It is expected that several convicted criminals may win appeals if their conviction was based
on this faulty evidence.
(e) Marks on Spent Cartridge Casings
• Firing pin marks left on the spent cartridge casings can also be used to identify a firearm
(Figure 9-5).
• Firing pin marks are impressions made on the bottom of the cartridge by the firing pin as it
strikes the bottom of the cartridge when the firearm is shot.
• Breechblock markings are another kind of mark left on spent cartridge casings. When the
firearm is shot, the explosive force pushes the bullet forward.
• At the same time, an equal and opposite force pushes the cartridge casing backward against
the breechblock, which prevents the cartridge from shooting toward the user as it recoils.
• Breechblock marks are produced as the cartridge casing moves backward and strikes the
breechblock. The markings are unique to the firearm and can be matched if the spent cartridge
casings are found (Figure 9-6).
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Figure 9-5: Depending on the firearm and Figure 9-6: Breech marks come in various
cartridge used, the fire pin mark appears on the forms, sometimes as parallel lines, circular
rim or the center of the spent cartridge. lines, or stippled at the bottom of the
cartridge casings.
• Other marks left on spent cartridge casings include extractor and ejector marks, which are
minute scratches produced as the cartridge is placed in the firing chamber (by the extractor)
and removed from the chamber after firing (by the ejector).
• These marks are produced only in semiautomatic and fully automatic weapons. In revolvers,
cartridges are hand-fed into the revolving cylinder and have to be removed by hand as well.
(f) Gunshot Residues
• Because all firearms explode gunpowder, they produce gunshot residues (GSR) when fired
(Figure 9-7).
• These residues are the traces of smoke and particles of unburned powder carried sideways from
the firearm by the expansion of gases as the bullet is fired.
• Gunshot residues containing nitrates can stick to the person holding the firearm and leave
evidence on the shooter.
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Figure 9-7: Gases are expelled from a firearm when it is fired and the gunpowder explodes.
• The amount of GSR decreases as the distance between firearm and victim increases.
Investigators look for the presence of GSR when attempting to recreate a crime scene.
• If someone fired a gun, GSR could be found on his or her hands or clothing. GSR can be
removed by washing, but chemical testing can often detect residue despite the attempted
removal.
• The distance between the weapon and the victim can be determined by examining the GSR
pattern on the body of a victim.
(g) Databases
• A database is a searchable collection of information stored in a computer system. Firearms
databases can be searched to match crime-scene evidence to registered weapons.
• Two important databases are the National Integrated Bullet Identification System (NIBIS),
which has computer files of ballistic markings of firearms used in previous crimes, and
Drugfire, an FBI database that focuses on cartridge casings.
• In 2000, these databases were merged to form the National Integrated Ballistics Network
(NIBIN).
(h) Digging deeper with Forensic science e-collection

• Some people believe that the development of a ballistic fingerprint database that catalogs the
rifling patterns of all registered guns could help solve future criminal investigations. Others
worry that this is an unnecessary infringement on the privacy of gun owners.
(i) Bullet Wounds
• Eyewitness accounts of a shooting are not always accurate. It is often helpful to examine bullet
wounds on victims to confirm or dispute a witness’s story.
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• For example, if someone claims that a victim was running away from a shooter at the time of
a shooting, an examination of the wounds on the body can confirm or dispute whether the
victim was indeed shot in the back.
• Determining which wound is the entrance wound and which is the exit wound is an important
step in determining what happened at a crime scene.
• Generally, entrance wounds are smaller than exit wounds, because the skin is somewhat elastic,
and it stretches when a bullet enters the body.
• Therefore, the size of the entry wound will be smaller than the bullet. Exit wounds are generally
larger, because as the bullet moves through the body, it may collect and carry body tissue and
bone with it.
• Another way of determining entry and exit wounds is to look at clues on the body near each
wound. For example, if the bullet penetrates clothing first, fibers may be embedded in the
wound pointing in the direction of penetration.
• Also, investigators can examine the presence of GSR, which is usually found only around
entrance wounds.
• Furthermore, if the bullet is fired when the muzzle is in contact with the skin, the hot gases
released from the muzzle flash may burn the skin, leaving a telltale mark.
• Bullets usually do not travel smoothly through a victim’s body. Bones, organs, and other
tissues bend their paths, causing a tumbling effect. The tumbling bullet may also tear a larger,
more irregular exit wound than expected.
• A bullet may ricochet off bone and do considerable internal damage before exiting. A bullet
may not exit the body at all.
• Several factors influence whether a discharged bullet will pass through a victim or remain
lodged somewhere in the body.
• For example, if the bullet has a high speed, it may have enough energy to pass directly through
the body, leaving both an entrance wound and an exit wound. High-speed bullets are more
likely to pass through a body than are low-velocity bullets.
• Small-caliber bullets, such as a 0.22 caliber, tend to lodge within the body, while larger caliber
bullets will pass through.
(j) The examination of suspect firearms
• The examination of any given firearm must be carried out in a planned, systematic and logical
fashion.
• There are actions that if carried out in the wrong order may destroy valuable information and
may even place the firearms examiner in danger of injury.
• For example, if a weapon is test-fired before the inside of its barrel has been inspected, this
will preclude the detection of small particles of material of potential evidential value (such as
gunshot residues) that were lodged within it prior to the test-firing.
• However, routine observations recorded during the examination of a firearm will typically
include:
1. Its type (e.g. shotgun, self-loading pistol);
2. Its condition as received (recorded with the aid of photographs, where practicable);
3. The number of cartridges that it (or its magazine) can hold;
4. Its manufacturer, make and model, bearing in mind that there have been instances in which:
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(a) unscrupulous gunsmiths have been known to produce counterfeit goods that are
designed to appear to be those of a well-reputed brand,
(b) guns designed to be able to fire only blanks have been adapted to fire projectiles,
(c) individual amateur gunsmiths have produced ‘home-made’ guns;
5. Its serial number (serial numbers are frequently removed by criminals but, in many such
cases, may still be rendered legible);
6. Its calibre (this information is normally embodied in the make and model of the gun
concerned; however, it is quite possible that the weapon concerned has been altered since
manufacture – this is particularly likely in the case of illegally reactivated guns that had
been previously legitimately deactivated and sold as non-firearms);
7. If it has a rifled barrel, the class characteristics of its rifling (e.g. the number of lands and
grooves and the direction of twist, i.e. whether the direction of the rifling spiral is right- or
left-handed);
8. If it has a smooth bore and is a shotgun, the degree of choke of the barrel(s) and whether
this degree of choke can be readily altered by the shooter;
9. Its weight (if required), overall length, barrel length and the distance from the muzzle to
the trigger(s);
10. The positions of the safety catch (where present) and any levers that allow the shooter to
select specific features of the gun (e.g. switch between selfloading and automatic modes of
firing).
• The examination of a given suspect firearm may help to answer a number of important
questions such as;
1. With whom or what has this firearm been in contact?
2. Could this firearm be responsible for firing the shots that were discharged at a given
shooting incident?
3. Could this firearm have been unintentionally discharged?
4. Could the intentional discharge of this firearm have caused unintentional injury?
5. Could this firearm have been used in the commission of an act of suicide?
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TOPIC 9: DOCUMENT ANALYSIS

• The term ‘questioned document’ is a wide-ranging term applicable to any document over
which there is some dispute or query and to which the skills of a document examiner may be
brought as part of the investigative process.
• Document examination is a very important and highly skilled area of forensic science,
requiring thorough training of its practitioners and access to suitably equipped laboratories.
• Some examples of typical questions addressed to forensic document examiners include;
1. Was the sample of questioned handwriting created by a particular person?
2. Is the signature forged?
3. Is the date that appears on the disputed document compatible with its age?
4. Has the receipt been altered?
5. Have the entries in this diary been written over a period of months or all on one occasion?
6. Were a number of different documents written by the same individual?
7. What writing appears beneath the obliterated portion of text?
8. What type of machine was used to produce the text of a questioned document?
9. Is the document genuine or counterfeit?
10. Can you tell us about the origin of this anonymous document?
• The key analysis aspects of questioned documents include; handwriting and signature
investigation, the examination of typed, word processed and photocopied documents, printed
documents and counterfeiting, and ink and paper analysis.
(a) Handwriting investigation

• The writing of each person is unique to him or her. Additionally, each piece of writing from a
given individual is in itself unique, and the writings of that individual vary over a natural range,
which is another feature of that person’s writing.
• As a consequence, handwriting can be used as a means of individual identification, provided
that sufficient quantities of specimen material (preferably non-request) are available for
comparison with the questioned handwriting
• Different basic types of handwriting are recognized; designated as block capitals (i.e. upper-
case unjoined writing), cursive writing (i.e. lower-case joined-up writing) and script (i.e.
lower-case unjoined writing).
• Two other terms namely connected writing and disconnected writing which forensic
handwriting experts usually consider to be synonymous with cursive writing and script
respectively.
• The normal handwriting of most individuals is somewhere between cursive writing and script.
• Handwriting experts will normally use the term ‘cursive writing’ to denote handwriting in
which the letters within words are predominantly joined.
• Conversely, the term ‘script’ is used for handwriting in which the majority of the letters within
words are not joined. Signatures are a specialized form of handwriting.
(b) Signature investigation
• Signatures differ significantly from bulk handwriting in that they are highly stylized portions
of writing – so much so that in some cases, they are completely illegible!
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• They are used as a means of personal identification and, as such, are produced on numerous
occasions, for signing cheques, letters, etc.
• This repeated usage means that their production becomes essentially automatic to the
individual.
• However, because of natural variation, a person’s signature is never identical on two separate
occasions. Signatures are often the subject of fraudulent reproduction (i.e. forgery).
• There are a number of handwriting characteristics associated with forged signatures that will
alert the experienced document examiner to the fact that they may not be genuine.
• A selection of these is given below (usually, more than one of the following signs are present):
1. ‘Shaky’ handwriting (apparent when viewed under magnification). This occurs when the
forger concentrates on copying the genuine signature very accurately by writing slowly
(thus resulting in a loss of fluency).
2. Unnatural pen lifts. This shows that the forger has paused to check progress.
3. Pen strokes with blunt ends where the pen has been lifted from the paper. This indicates
that the pen strokes of the writer have been made slowly and deliberately, while in fluent
writing, such pen stroke ends tend to be tapered. Low-power microscopy is needed to view
this particular feature.
4. Evidence of retouching. This indicates that the forger has attempted to patch up ‘less good’
parts of the signature in an effort to make it more realistic.
5. Difference in scale. The writing is noticeably smaller or bigger than the genuine writing.
6. Incorrect proportioning of the letters.
7. Unnatural similarity between two (or more) signatures. (Such close correspondence
between signatures would not occur if the signatures were genuine because of the range of
natural variation shown in an individual’s normal handwriting.)
8. The positioning of the signature relative to the rest of the document.
(c) Typed, word-processed and photocopied documents
• The evolution in the technological production and reproduction of documents within the office
environment has necessitated a concomitant development in the expertise of forensic
document examiners.
• The questions most frequently asked of them are likely to concern the type of machine used to
produce a questioned document (and, if possible, its make and model) and whether a particular
suspect machine was used to produce a specific questioned document or documents.
• Typewriters may be either manually or electrically operated. Manual typewriters have
fixed typebars, which means that the style of their typeface cannot be changed except by a
typewriter mechanic.
• Single-element electric typewriters differ from manual typewriters in that their typeface is not
fixed but carried on a single element that can easily be replaced.
• Computer-based word-processor systems have almost totally eclipsed typewriters as the
means of document production.
• In essence, keyboard operators use word-processor programs in order to compose documents,
which are then usually stored electronically on computer file.
• These files may be printed out as and when required, using any of the several types of computer
printers available on the market.
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• Modern photocopiers use plain paper. Plain paper photocopiers are extensively used in the
office workplace for the reproduction of documents, as well as being widely available to the
general public, for example in shops (newsagents, post offices, etc.) and libraries.
• They operate on the same principle as the laser printer, using light to facilitate the creation of
an image of the original document.
• The forensic document examiner may be able to identify the model of photocopier used to
produce a disputed document by the examination of certain features of the photocopy.
• Such features may include any characteristic marks caused by the mechanism used to handle
the paper during copying, and the morphology and chemical composition of the toner used.
(d) Printed documents
• There are a wide variety of printed documents in common use, each fulfilling a particular
function.
• Examples include banknotes, passports, cheque books, vehicle registration documents,
MOT test certificates and tax discs.
• Each of these types has an intrinsic value, for example as a means of personal identification,
as proof of ownership, or in monetary terms. As a result, many types of printed documents are
prone to fraudulent reproduction.
• Four main types of traditional conventional printing methods namely; screen printing,
letterpress (also known as relief printing), gravure (also known as intaglio) and lithography
involves the use of pressure to transfer ink to create the image on the paper.
• In contrast, modern computer printing technologies involve minimal or no contact. These are
termed non-impact printing methods and include ink-jet and laser printing.
(e) The analysis of handwriting inks
• A visual examination of the writing on a document, using low powered microscopy, may
provide general information on the type of ink, and therefore the type of writing instrument,
used.
• For example, text written using ballpoint ink is readily identifiable, being thick and glossy in
appearance, often with characteristic striation marks. These striations originate from
incomplete inking of the ball that is used to transfer the ink to the page.
• Much of the work carried out by forensic document examiners in this area therefore involves
a comparison of the inks found on a particular document to establish whether they are different.
• The discovery of more than one ink on a single document might suggest that it has been altered
at some stage.
• The tests used for ink analysis fall into two broad categories: non-destructive and destructive.
• Non-destructive tests are used preferentially before destructive ones and mainly concern the
examination of the inked text under different lighting conditions.
• Furthermore, infrared and Raman microscopes have now been developed that make it possible
to obtain both infrared and Raman spectra of inks in situ on documents.
• There are two chromatographic methods that are suitable for the separation of dyes in ink
samples – high-performance liquid chromatography (HPLC) and thin-layer chromatography
(TLC) and the latter is frequently used
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(f) Paper analysis

• Forensic document examiners skilled in paper analysis involve a comparison of the
characteristics of two (or more) pieces of paper to ascertain whether or not they may have a
common origin.
• The properties of paper are determined by many variables, such as the type of raw fibres used,
the choice of sizing agent and the decision whether or not to bleach the fibres during the
various stages of the manufacturing process.
• There are basically two categories of tests available, namely non-destructive and destructive.
Non-destructive tests are applied first, as these do not entail any damage to the documents.
• Such tests usually involve a visual comparison of features, such as the colour, size and shape
of the sheets of paper, while thickness may be measured using a micrometer.
• Another non-destructive test that may be applied in this regard is a comparison of the
fluorescent properties of the pieces of paper when viewed under ultraviolet (UV) light.
• Destructive tests can yield information on a number of different properties of the paper, which
can be used for comparative purposes, including:
1. The types of raw fibre used (established by microscopic examination);
2. The type of pre-treatment used to prepare the pulp, whether chemical or mechanical;
3. The nature of the surface coating (established by X-ray powder diffraction).
• Dating of paper may yield valuable information about the authenticity of disputed documents.
In this respect, the watermarks used by paper manufacturers are particularly useful.
• Watermarks contain fewer fibres per unit area than the rest of the sheet of paper and, as such,
are usually visible when the paper is held up to the light (
(g) Tears, folds, holes, obliterations, erasures and indentations

• A number of features can yield important information concerning the history, origin and/or
authenticity of questioned documents. These are tears, folds, holes, obliterations, erasures and
indentations.
• Tears: It is possible to demonstrate that two or more pieces of paper were originally part of a
larger sheet by mechanically fitting the torn pieces together.
• Some types of document are designed to incorporate a line of weakness, such as a series of
short cuts in the paper or a line of perforations (either elliptical or circular), so that part of the
document is more easily torn off.
• A common example is the chequebook, where cheques are torn out when required, each
leaving behind a stub in the chequebook.
• Such tears are often irregular in nature, leaving pieces of paper of uneven length on either
side of the tear.
• In these cases, it may be possible to make a match between two parts of a document torn
along perforations.
• Other examples include books of matches, as may be found in arson cases, and perforated
sheets of stamps.
• Folds: the presence of folds on a document may yield significant information about the order
in which ink lines have been made and, consequently, the order of writing.
• This is possible because the nature of the ink mark at the point of fold is different depending
on whether it was made before or after the document was folded.
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• Essentially, those ink marks made after folding contain comparatively more ink, partly because
the surface of the paper acquires damage along the fold line.
• Holes present in documents are often the result of stapling, a method used for fastening two
or more separate sheets of paper together, usually in the vicinity of the top left- or right-hand
corner.
• In stapling, the open ends of the staple are forced to pierce through the sheets of paper and
then bent towards each other under the final sheet, thus holding them all together.
• The position, number and even appearance of staple holes may provide useful information to
the forensic document examiner.
• A close match between the position and size of staple holes on two, or more, separate sheets
of paper is consistent with their having been previously attached together.
• If patterns of distortion around the staple holes on separate sheets are also similar, then this
provides a further indication that they were once held to one another.
• Obliterations: deliberate obliteration may have been made by the application of certain
substances such as the same ink that was used to create the original text, or a different
substance such as another ink or a correction fluid.
• Different approaches may be taken to decipher the writing hidden beneath the obliterating
medium.
• In some cases, it may be possible to interpret the original writing by simply examining the
reverse of the document- writing apparent will appear as a mirror image.
• Examination under specialist lighting conditions will often enable the original writing to be
read.
• There are several pieces of equipment commercially available for this purpose, such as the
Video Spectral Comparator (VSC) built by Foster & Freeman Ltd and the Docucenter 3000
made by the Swiss firm Projectina AG.
• Among the features of this type of apparatus are a variety of light sources, including different
wavelength bands of visible light, ultraviolet and infrared in reflectance mode, and transmitted
white light.
• These features can be used to help distinguish between the different types of inks, if more than
one is present, and can also be used to reveal writing obscured by correction fluid.
• In the case of typewritten material obliterated by correction fluid, the application of a suitable
solvent may be successful.
• This renders the dried fluid translucent, for a brief period of time, thus enabling the typewriting
underneath to be read.
• Erasures: erasures are interpreted as those instances where writing is actually removed from
a document, using either mechanical or chemical means.
• The mechanical erasure of writing may involve the use of a suitably abrasive medium, for
example hard rubber, or the removal of slivers of paper by means of a sharp instrument such
as a knife.
• Chemical methods of erasure include the application of bleaching agents, which convert the
dye into colourless materials, and the use of certain solvents.
• Whatever the method of erasure, it may be possible to detect minute traces of the original
writing material, by, for example, inducing them to luminesce.
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• Indentations: this phenomenon commonly occurs when an individual writes on one document
while it is resting on another.
• In a similar manner, if, for example, a diary or pad of paper were used, indentations of the
handwriting might be apparent, albeit with increasing faintness, on several subsequent pages.
• As well as direct impressions resulting from the act of writing itself, ‘secondary impressions’
may be created under certain circumstances.
• For example, impressions of writing may be transferred by contact between documents when
they are stored together in close proximity.
• The method selected for the detection of indented writing is influenced by the depth to which
the impression has been made.
• If the indented writing is sufficiently deep, it may be possible to read what is written by
illuminating the document with an oblique light source.
• However, a more sensitive method, suitable for shallower impressions, is the application of
the electrostatic detection apparatus (ESDA).
• It is noteworthy that ESDA does not always reveal the presence of some deep impressions that
are nonetheless visible in oblique lighting. Thus, these two methods complement each other.
TOPIC 10: FORENSIC BIOLOGY

(a) Analysis of hair
• A hair is a fibre that grows out of a hair follicle in the skin of a mammal. At one end of any
one hair is its root.
• Unless the hair has been pulled out, this is found within the skin. At the other end is the hair’s
tip. The hair shaft is the main portion of the hair that lies between the root and the tip.
• A longitudinal view of a mature hair shaft, as seen using a light microscope, shows it to contain
up to three main morphological features, namely:
1. The cuticle (the outer layer of the hair shaft, made up of tough, flattened cells called scale
cells);
2. The cortex (made up of spindle-shaped (i.e. fusiform) cortical cells, which are cemented
together);
3. The medulla (made up of collapsed cells and intercellular and intracellular air-filled voids).
• The morphological study of hair has revealed a number of forensically valuable observations,
in particular those listed below:
1. There are species-to-species variations in the structural detail observable within hair. For
example, a fundamental distinction of this type is that between human and non-human hair
in terms of colour, medulla width, medulla appearance, pigment granules distribution and
scale pattern.
2. Deduce from which part of the body a given human hair originated (e.g. scalp hair is
morphologically distinguishable from pubic hair).
3. Human hair morphology carries indications of racial type.
4. The root of a human scalp hair that was pulled out while the hair was actively growing (i.e.
in the anagen phase of the growth cycle) is characteristically flame shaped broken, and
may have follicular material attached to it. In contrast, a human scalp hair that was detached
from the skin after the hair had ceased growing (i.e. it was in the telogen phase of the
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growth cycle when it was removed) has a root with a characteristic club-like appearance.
Hair in the anagen phase is much harder to pull out of the scalp than that in the telogen
phase.
5. Morphology can reveal differences between hairs taken from the same body location on
different individual humans.
• The main morphological points of comparison used to evaluate associative evidence based on
human hair include:
1. the colour, length, waviness and diameter of each individual hair;
2. the size, shape, density and distribution of observable pigment granules in each hair shaft;
3. cosmetic alteration of each hair;
4. damage to each hair, including any caused by disease or dietary deficiency;
5. the presence or absence of nits or fungal infection;
6. the shape of the cross-section of the hair shaft.
• If the roots of such hairs have cells from the hair follicles still adhered to them, there may be
sufficient nuclear DNA available for the individual to be identified through DNA profiling.
• Although it is possible to extract DNA from the hair shaft irrespective of whether or not the
hair was actively growing when it was detached from the skin, this is rarely done.
• This is because hair shaft DNA is mitochondrial and this is of lower evidential value than
nuclear DNA.
• Finally, it is worth noting that the recent development of Low Copy Number DNA techniques
means that it is now possible to obtain a DNA profile from the nuclei of the few skin cells that
may be adhered to the hair shaft as a consequence, for example, of dandruff.
• In many cases, the likelihood of the transfer of hairs is heightened by violent contact such as
in cases of violent crime, such as assault, rape or murder.
• Presence of hairs in a crime scene may be instrumental in the furtherance of an investigation.
For example, the presence of several similar, long, scalp hairs of human origin at the scene of
a crime could strongly suggest that a person with, or who had, long hair has at some time been
present at that scene.
• This information may prove to be important in assisting the police in their search for a victim
or perpetrator of the crime or an eyewitness of the incident.
(b) Analysis of fibre
• The term fibre (spelt fiber in the United States) is used to denote any solid object that is thin,
flexible and elongate, having a high length to transverse cross-sectional area ratio.
• Fibres of forensic interest can be classified on the basis of their origin and composition.
• These include hairs, especially those of human origin, and those man-made and natural fibres
that are formed into textile products that are used in the manufacture of clothing and household
fabrics.
• Many of these common fibres are readily transferred from one object to another during
physical contact.
• Interestingly, in most environmental conditions, these common types of fibre are resistant to
biological, physical and chemical degradation and therefore persist intact for long periods of
time.
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• Furthermore, their examination provides multiple points of comparison that can enable them
to be identified to the class level. For example, it is possible to determine the type of polymer
(e.g. polyester) from which a particular man-made fibre is formed.
• The achievement of this class level of identification, coupled with the comparison of the
characteristics exhibited by a questioned fibre obtained from an incident scene and a control
fibre (such as that taken from the belongings of a suspect), can provide useful information.
• Importantly, such information is frequently enough to enable the fibre examiner involved to
conclude that the two fibres are either different or, alternatively, sufficiently similar that they
may have originated from the same source.
• The forensic examination of fibres is primarily carried out in order to identify the individual
fibre types (i.e. whether human scalp hair, nylon textile fibre, etc.) and to provide descriptions
of characteristics that can be used as points of comparison between the fibres of interest.
Figure 11.1 A classification of fibres of interest to forensic science

• Much of this work is based on microscopic examination. However, the starting point is the
inspection of the macroscopic features of the specimen, firstly with the unaided eye and then
using a low-power stereomicroscope.
A. The recovery of trace-level fibres
• Any technique used to obtain trace-level fibres should ideally sample all of the types of fibre
that have been transferred to the object being sampled without taking fibres from the object
itself.
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• There are a number of approaches that can be adopted in order to attempt to achieve these
objectives. These include;
1. Hand picking with tweezers.
2. Using a vacuum cleaner fitted with a specialist attachment that directs the stream of air
onto a paper or fabric filter.
3. Scraping.
4. Combing (of hair).
5. Making tape lifts.
• All of the approaches have advantages. However, for the majority of work, tape lifting is
usually the method of choice.
• Once a target fibre has been identified, the dissection of the tape and the application of a small
quantity of solvent (e.g. xylene) to dissolve the tape’s adhesive can enable it to be removed.
• The target fibre can then be mounted on a microscope slide for subsequent examination.
B. The fibre characteristics that are examined
• There are a number of techniques that can be used to characterize fibres and to produce
additional points of comparison. Among these are the following:
1. Forensic examination of fibres using light microscopy describes their morphological
features and optical properties (e.g. colour, any fluorescence under ultraviolet light and
birefringence).
2. Dye extraction from questioned and control fibres, followed by thin-layer chromatography.
3. Microchemical tests that can be carried out on the dyes and/or pigments of small sections
of fibre that have been placed on microscope slides beneath cover slips. Under these
circumstances, the fibre sections can be exposed to liquid reagents (such as bleach (sodium
hypochlorite solution) or concentrated sulphuric acid) by placing a small drop of the
reagent concerned on an edge of the cover slip. Capillary action will then draw the reagent
under the cover slip and around the fibre. Any observable colour changes can then be
viewed with a compound light microscope and used as points of comparison.
4. Scanning electron microscopy (SEM), which is particularly good at providing details of
surface morphology.
5. Melting point determination, which is applicable to many man-made fibres.
6. Pyrolysis-gas chromatography of man-made fibres.
7. The ashing of natural fibres followed by microscopic examination of the residues.
• Evidence of any kind must be evaluated, and this is especially important for fibres because
they are so plentiful in the environment.
• The value of fibre evidence in a crime investigation depends on its potential uniqueness. For
instance, a white cotton fiber will have less value than an angora fibre, because cotton is so
common. A forensic scientist will ask questions about the following:
1. Type of fibre: What is the composition of the fiber? How common or rare? What suspects
or victims or part of the crime scene had this type of fiber on them?
2. Fibre color: Do the fibers from the suspect’s clothes match the color found in the victim’s
house? Is the type of dye the same?
3. Number of fibers found: How many fibers were found—one or hundreds? More fibers
suggest possible violence or a longer period of contact.
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4. Where the fiber was found: How close can you place the suspect to the scene of the
crime—in the house, or close to a victim’s body?
5. Textile the fiber originated from: Are these carpet fibers, or upholstery from a car?
6. Multiple fiber transfers: Is there only one type of fiber transferred at the crime scene? Or
are there fibers from numerous sources from carpets and clothes and bedding? More
sources suggest longer contact or possible violence.
7. Type of crime committed: Was the crime violent, a break-and-enter, a kidnapping? Each
type of crime has an expected pattern of contact between suspect, victim, and crime scene
that will be reflected in the transfer of fibers.
8. Time between crime and discovery of fiber: How long ago did the transfer take place—
an hour, a day, a week? Unless the fiber location is undisturbed (such as a bagged jacket
or locked room), the value of found fiber is greatly reduced with the passage of time
because fibers will be expected to fall off, or fibers not related to the crime can be picked
up.
C. Analysis of biological fluids

(a) Blood Spatter Analysis
• When a wound is inflicted and blood leaves the body, a blood-spatter pattern may be created
which constitute a grouping of bloodstains forming a pattern. This pattern can help reconstruct
the series of events surrounding a shooting, stabbing, or beating.
• Blood-spatter patterns can help determine the manner of death, based on the blood velocity.
Instructions on blood-spatter analysis are provided within each activity.
• Blood is a thick mixture of blood cells and plasma. When a person is injured and is bleeding,
gravity acts on blood, pulling it downward toward the ground. The blood droplet has a tendency
to become longer than it is wide as a result of gravity.
• Blood is cohesive. This means that the blood mixture is attracted to similar blood mixtures and
tends to stick together and not separate as it falls.
• The effect of the downward force of gravity combined with the cohesive force of the blood
results in a net effect on the blood droplet as it falls. Thus, the blood maintains a circular or
round appearance.
• If any of the blood does overcome cohesion and separate from the main droplet of blood, it
will form small secondary droplets known as satellites.
• If blood is dropped onto a smooth surface, such as glass or marble, the edge of the blood drop
appears smooth and circular.
• However, if the blood lands on a porous surface, such as wood or ceiling tile, then the edge of
the drop of blood may form small spikes or extensions. Notice that spikes are still connected
to the main droplet of blood, whereas satellites are totally separated.
• In 1902, Dr. John Glaister first described the six patterns into which blood spatters could be
classified. They include:
1. Blood falling directly to the floor at a 90-degree angle will produce circular drops, with
secondary satellites being more produced if the surface hit is textured. This is known as a
passive fall.
2. Arterial spurts or gushes typically found on walls or ceilings are caused by the pumping
action of the heart.
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3. Splashes are shaped like exclamation points. The shape and position of the spatter pattern
can help locate the position of the victim at the time of the attack.
4. Smears are left by a bleeding victim depositing blood as he or she touches or brushes
against a wall or furniture.
5. Trails of blood can be left by a bleeding victim as he or she moves from one location to
another. The droplets could be round or smeared or even appear as spurts.
6. Pools of blood form around a victim who is bleeding heavily and remains in one place. If
the bleeding victim moves to another location, there may appear to be droplets or smearing
connecting the first location with a second.
• Bloodstain processing involves extraction critical information such as:
1. Confirm the stain is blood. There are two tests:
(a) Kastle-Meyer test. If blood is present, a dark pink color is produced.
(b) Leukomalachite green. This chemical undergoes a color change, producing a green
color in the presence of blood.
2. Confirm the blood is human using an ELISA test (Enzyme Linked Immunosorbent
Assay) which involves an antibody–protein reaction.
3. Determine blood type by examining antigen–antibody reactions.
4. Determine if it is a Transfer bloodstain. Transfer bloodstains are those that have been
deposited on surfaces as a result of direct contact with objects contaminated with wet blood.
• Bloodstain pattern analysis can be used to:
1. To help establish the events that took place during the course of a violent attack.
2. To help establish the probable sequence of events that occurred.
3. Determine the location of any bloodstains present at a crime scene and the quantification
of the amount of blood involved.
4. Refute or corroborate a particular version of events as given by a suspect or witness using
blood pattern evidence.
5. Identify different types of blood present in a scene using DNA profiling.
A. Presumptive tests for blood
• Presumptive Tests are used to tell if a sample is blood (and not of some other substance, such
as ink, rust or chocolate) before other, more complicated, blood-specific tests are carried out.
• The presumptive tests used for blood are based on the ability of the haemoglobin present in
red blood cells to catalyse the oxidation of certain reagents.
• Other presumptive tests can tell if a sample is human or primate blood.
• This is important to avoid wasting time trying to purify DNA from red paint and meat juices.
• Take a small rubbing from the stain, react it with chemicals and look for a color change.
1) Color tests using phenolphthalein (Kastle-Meyer color test)
• We use phenolphthalein for color tests
• When phenolphthalein is mixed with the blood’s hemoglobin, it produces a deep pink color
• This is known as Kastle-Meyer color test which is indicative of blood. Hemoglobin causes a
deep pink color
• Chemical phenophthalein turns dried blood into pink showing evidence of blood.
• It will also turn pink if put on potatoes and horseradish
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2) Luminol
• The luminol test is used to search out trace amounts of blood located at crime scenes. Produces
light (luminescence) in a darkened area from bonding with hemoglobin left behind from a
crime scene.
• Luminol sprayed on blood, will fluoresce (glow) blue when the lights are turned off. It’s useful
in determining if blood is present. It takes 5 seconds to read
• It will detect blood that has been diluted up to 300,000 times. Tiny particles of blood will cling
to most surfaces for years and this test can be used on old blood
• It destroys the blood for further serological testing but it does not mess up DNA tests
• Hidden blood spatter patterns can help investigators locate the point of attack and even what
sort of weapon was used (a bullet makes blood splatter very differently than a knife does).
• Luminol may also reveal faint bloody shoe prints, which gives investigators valuable
information about the assailant and what he or she did after the attack
• Once it has been determined that the bloodstain is of human origin, an effort must be made to
associate or dissociate the stain with a particular individual before DNA analysis
3) Hemastix
• Reagent strips with TMB ends (Tetramethylbenzadine). TMB changes from orange to green
when it comes into contact with blood
• Used for occult blood in stool
• It is somewhat quantitative and very sensitive. However, TMB is dangerous
• Hemastix strips are moistened with distilled water and put on blood stains.
• If it turns green, it is blood.
4) Gel diffusion
• Antigens and antibodies will move towards each other when put on a gel plate.
• Each is put in a hole opposite to the other.
• If the blood is human, they will form a line between the two holes.
• Once the blood has been determined to be human, DNA testing takes over.
B. Serological tests for blood
• The term serology is used to describe a broad scope of laboratory tests that use specific antigen
and serum antibody reactions.
• If presumptive testing indicates that a particular stain is composed of blood, the next logical
step is to ascertain whether that blood is of human origin. This can be done by:
1. Using the precipitin serological test to identify the presence of proteins specific to humans;
2. Analyzing for DNA sequences specific to humans.
• The precipitin test may be applied to bloodstains in a number of different ways. For example,
it may be conducted in a capillary tube, with a layer of human antiserum (i.e. serum containing
antibodies specific for human antigens) overlain by a layer containing an extract of the
bloodstain under investigation.
• The formation of a cloudy precipitate at the interface between the two layers indicates a
positive result for human blood.
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• In another method, known as cross-over electrophoresis, a gel-coated slide containing twin

wells is used. A liquid extract of the bloodstain is placed in one depression, while human
antiserum is placed in the other.
• The application of an electric current to the slide induces the antibodies (from the antiserum)
and the antigens (from the blood sample) to move towards each other.
• If a line of precipitation forms where the two meet, then the bloodstain is human in origin
(b) Forensic characterization of semen
• Many sex crimes are committed each day. Sperm have DNA which can be well preserved if
dried on cloth.
• Forensic scientists have to sift through ALL of the stains in underwear, sheets, carpets, etc.
• The first part of testing begins with finding the stain. The second part is actually testing it.
• Semen is a fluid of complex composition, produced by the male sex organs. There is a cellular
component, spermatozoa, and a fluid component, seminal plasma
1) Seminal Plasma
• It is composed of salts, sugars, lipids, enzymes, nutrients, proteins, hormones, basic amines
(spermine), P30, flavins
• An example of enzyme is Acid Phosphatase
• P30 is a prostate specific protein used in prostate cancer tests
• Flavins components originate from several sources, including seminal vesicles and the prostate
gland
2) Sperm Cells
• Sperm are the male reproductive cells. Each consists of a head, tail and midpiece
• In humans, the head is a tiny disc, about 4.5 um long and 2.5 um wide. The tail is about 40 um
long, and is rapidly lost in ejaculate
• Normal males release 250-600 million spermatozoa each time.
• Dogs have similarly shaped sperm, but are about three times larger than human sperm. Other
animals have differently shaped sperm
3) Presumptive tests for Semen
• Semen stains fluorescent under UV light. It is common practice to visually assess items of
evidence under UV light to locate possible semen stains
• The intensity of the fluorescence can be affected by the substrate, concentration of the stain,
and other body fluids
• Semen can be identified on crime scene or in lab by Evidence Technician
• Apart from semen fibers, coffee, food, detergent, most organic stains also fluoresces
A. Acid Phosphate test
• There is 400X’s more acid phosphate in semen than in any other body fluid and several
chemicals are able to detect acid phosphate.
• Alpha naphthylphosphate and Fast Blue B dye turn purple when they come in contact with
semen and are mainly used on fabrics and surfaces
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• If you are doing a big area (carpet, sheets), you use 4-methyl umbelliferyl phosphate (MUD)
which will glow when it comes in contact with semen. A reaction time of less than 30 seconds
means you have found semen, not something else.
• While AP is detected in high concentrations in semen, it can also be detected in other body
fluids
• False positives includes Vaginal acid phosphatase, Fecal material, Plant matter,
Spermicides (orange), Some feminine hygiene products etc
B. Microscopic examination of semen

• Sperms are very easy to identify under a microscope. Spermatozoa have a head and a long,
thin tail.
• Put the sample in a small amount of distilled water and stir. Some of the spermatozoa will go
into the water. A drop of this water will be dried, stained and looked at under 400X power.
• Most common staining method is Kernechtrot picroindigocarmine stain- Also called
Christmas Tree Stain, it is either prepared or commercially supplied
• There are problems collecting the sperm for testing since they bind very tightly to fabrics, so
it is hard to get a sample.
• They are also very brittle when they dry. The male may have a low sperm count or no sperm
count (vasectomy).
(c) Forensic characterization of saliva
• Saliva is a colorless fluid secreted by 3 glands in the mouth; sublingual, submandibular, and
parotid.
• Saliva from parotid glands contain amylases enzymes, which aid in the digestion of
carbohydrates. Saliva is composed of electrolytes, enzymes, mucus etc.
A. Screening Tests for saliva
• Screening for saliva is based on detection of high levels of amylase in the sample. It is not a
confirmatory test since amylase is found in other body fluids such as serum, urine, sweat, lip
mucous, semen, feces, etc.
• The concentration of amylase in saliva is variable among individual; if amylase is not detected
in a sample it does not mean saliva is not present
• UV light can be used to aid in locating saliva stains
• The intensity of the fluorescence can be affected by the substrate, concentration of the stain,
and other body fluids. Saliva does not fluoresce as intensely as semen
B. Amylase
• One of the earliest tests for amylase was the starch-iodine test. Iodine solutions cause starch
to turn a deep blue color. Amylase is a starch hydrolyzing enzyme.
• The presence of amylase causes the disappearance of the blue color (due to hydrolysis of the
starch) and can be used an indicator for the presence of amylase
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KENYATTA UNIVERSITY

DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY

(b) Determining the Value of Evidence

3) Footwear and tire track evidence

5) Locate and collect latent fingerprints

TOPIC 2: BIOCHEMICALLY SIGNIFICANT COMPOUNDS

No of C Category Moiety Examples

Thus sugars are given D or L designation depending on whether their configuration

(a) D- and L-glucose; (b) D- and L-fructose

6. Symmetry, asymmetry & chirality

α-D-Glucopyranosyl -(1→2)- β- fructofuranoside

8. “Boat” and “Chair” conformations

β-D-Glucose (chair form) β-D-Glucose (boat form)

Sucrose (Glucose-fructose) Lactose (Galactose-glucose) Maltose (Glucose-glucose)

3. Liquid or Solid State at Room Temperatures

Some of the omega (ω ) fatty acids

Fatty acid Structure Source

[3] Classification of proteins

(d) Nucleic acids

50×A260=concentration of DNA sample (µgml−1)

• Contaminants may also be identified by scanning UV spectrophotometry from 200 nm to 300

Homogenise Cells/Tissues Cellular Lysis Chelating Agents

Redissolve DNA Alcohol Precipitation Phenol Extraction

• Cryopreservation or formalin-paraffin fixation is often difficult or impossible under field

[2] Isolation of RNA

RNA solvents Proteinase Agents

Redissolve RNA Alcohol Precipitation Phenol Extraction

• It is possible to check the integrity of an RNA extract by analysing it by agarose gel

Figure 3.3: Affinity chromatography of poly(A)+RNA.

[3] Techniques used in DNA Extraction

TOPIC 4: PROTEIN ISOLATION AND PURIFICATION METHODS

E1 = no. of (Tyr) × Ext (Tyr) + no. of (Trp) × Ext (Trp)

E2 = no. of (Tyr) × Ext (Tyr) + no. of (Trp) × Ext (Trp)

Aliphatic index = {X (Ala) + 𝑎 } × { X (Val) + 𝑏} × {X (Ile) + X (Leu)}

• An increasing positive score indicates greater hydrophobicity.

(a) Edman degradation reaction

(b) Mass spectrometry

• In order to evaluate the progress of purification, a convenient assay procedure—based on

Procedure: Separation Method Property

C. Characterization of the isolated proteins

Property of Proteins Technique

(a) Introduction to chromatography

• Where TB is the retention time of component B, TA is the retention time of component A, wA

(a) Paper chromatography

Diagrammatic representation of TLC

• The following are some common uses of Thin-Layer Chromatography:

Diagram of a gel-filtration process

(i) Affinity Chromatography

2. Capillary electrophoresis (CE): Electrophoresis is done in a buffer filled, narrow-bore

Isoelectric focusing. This technique separates Two-dimensional electrophoresis.

TOPIC 5: PROPERTIES OF ANTIBODIES

(a) Antibody Class Functions

Summary of Immunoglobulins structures

Organism Antigens Antibodies

TOPIC 6: WESTERN BLOT TECHNIQUE

Western blotting work flow

(c) Medical diagnostic applications

TOPIC 7: ANALYSIS OF FIRE AND EXPLOSIONS

3. The establishment of legal liabilities associated with the fire.

• In order to be an explosive, a material must be capable of propagating the explosive chemical

(g) Patterns of explosion damage

TOPIC 8: ANALYSIS OF FIREARMS

A. History of Gunpowder and Firearms