CLASS RULES

Sophia J. Pangilinan, RMT

1. Be punctual
2. Be respectful to each and everyone
3. Strictly no cheating allowed
4. Change your zoom name
(e.g. Pangilinan, Sophia J. (3-YB-1A)
 TIPS
Your mind is a fertile soil.
Time is gold. Use it wisely.
Be responsible. Know your
priorities.
Patience and hardwork.
PRAY! MOST IMPORTANT.
POLYMERASE
CHAIN
REACTION
(PCR)
Sophia J. Pangilinan, RMT
 THREE IMPORTANT STEPS:
THERMAL CYCLER

1. DENATURATION (94 °C): the double

stranded DNA separates
2. ANNEALING: The preferred Tm temperature
is between 55 °C and 72 °C, and the ideal
temperature is between 62 °C and 65 °C
primers anneal to the single strand
 THREE IMPORTANT STEPS:
THERMAL CYCLER

3. EXTENSION (72 °C): uses Taq polymerase in

order to synthesize a new strand of DNA
The following are the things to consider in
designing a primer:

1. Primer specificity

2. Primer length: Primer length is typically 18-30

nucleotides
3. Nucleic acid content

4. Melting temperature or Tm
Most important consideration
The preferred Tm temperature is between
55 °C and 72 °C, and the ideal temperature
is between 62 °C and 65 °C
 DNA
TECHNOLOGY

Sophia J. Pangilinan, RMT

Nucleic acid sequencing (partial or whole
genome) of hundreds of species has provided
great amount of data for analysis and
comparison that gave rise to genomics and
advancements in biotechnology and medicine.
The completion of the Human Genome Project
(HGP) in 2003 has led to the advancement of
genetics and medicine and other related
sciences
GENOME ANNOTATION LEVELS
Interpreting DNA/nucleic acid sequences is
called DNA annotation.

DNA annotation or genome annotation is the

process of identifying functional elements along
the sequence of a genome, thus giving meaning
to it by identifying a possible function
a. Nucleotide-level annotation
It identifies the physical location of DNA
sequences to determine where
components, such as genes, RNAs, and
repetitive elements, are located.
This is the basic level of genome
annotation.
b. Protein-level annotation
This level of annotation tries to determine the
function of each gene by identifying its protein
product and its function.

c. Process-level annotation
The goal of the process-level annotation is to
identify the pathways and processes in which
different genes interact, assembling an efficient
functional annotation.
NUCLEOTIDE-LEVEL ANNOTATION TOOLS

a. Genome browser
UCSC Genome browser
b. Genome Alignment and Assembly Tools
NCBI Assembly Viewer
c. Sequence Alignment Tool
BLAST:
Basic Local Alignment Search Tool
is a computational tool that finds
the regions of similarity between
biological sequences
Moreover, the following terms are
important in analyzing BLAST results

• Homology
It refers to a similarity attributed to
descent from a common ancestor.
• Similarity
It refers to the extent to which nucleotide or
protein sequences are related.
Furthermore, very high similarities
(70%-99%) means the sequences can be
homologous sequences, but the most
important indication of homologous genes
is similarity in function
• Identity
It refers to the extent to which two
(nucleotide or amino acid) sequences are
invariant. Identity percentage,
specifically 100%, during the pairwise
alignment in BLAST is an indication that
the two sequences are identical
Conservation
it refers to the changes at a specific position of an
amino acid (less commonly, DNA) or sequence
that preserves the physico-chemical properties of
the original residue

a. Orthologous genes - These are homologous

sequences in different species that arose from a
common ancestral gene during speciation
b. Paralogous genes - These are homologous
sequences within a single species that arose by
gene duplication
RECOMBINANT
DNA
TECHNOLOGY
Sophia J. Pangilinan, RMT
Recombinant DNA is a technology that
involves inserting a human gene into the
genetic material of a common bacterium
through a vector, the plasmid. Through
this biotechnology, the "recombinant"
organism can produce the protein
encoded by the human gene
Recombinant DNA technology is divided into
three basic steps:

1. Amplification/isolation of the desired gene of

interest using a well-designed primer
2. Digestion of original plasmid and ligation of
the gene of interest to the vector/plasmid
3. Transformation of the bacteria to have the
recombinant DNA
AMPLIFICATION/ISOLATION OF THE DESIRED
GENE OF INTEREST USING A WELL-DESIGNED
PRIMER

Gene amplification/gene isolation requires

polymerase chain reaction (PCR). For PCR to be
efficient, there should be well-designed DNA
primers
 DIGESTION AND LIGATION OF THE GENE OF
INTEREST IN THE PLASMID

a. Restriction enzyme
are endonucleases that recognize short DNA
sequences and cleave double-stranded DNA at
specific sites within or adjacent to the
recognition sequences
b. DNA Ligase
is a DNA-joining enzyme
In recombinant DNA technology, the
gene of interest must be inserted to the
vector, plasmid
For ligation to occur, two pieces of DNA
must have matching ends; then, ligase
link them to form a single molecule of
DNA
TRANSFORMATION OF THE BACTERIA TO
HAVE THE RECOMBINANT DNA

Transformation is the process that

involves the uptake of the naked DNA
from the
surround medium by competent cells
RIBONUCLEIC
ACID

Sophia J. Pangilinan, RMT

 Codec Pro Fat

DNA RNA PROTEIN

Replication Transcription Translation
RNA VS. DNA
DEOXYRIBONUCLEIC
RIBONUCLEIC

ACID
ACID

Double-stranded Single-stranded
Base pairs Base pairs
Adenine:Thymine Adenine:Uracil
Cytosine:Guanine Cytosine:Guanine
Deoxyribose sugar Ribose sugar
DEOXYRIBONUCLEIC
RIBONUCLEIC ACID
ACID

PURINE PURINE
Adenine Adenine
Guanine Guanine
PYRIMIDINE PYRIMIDINE
Cytosine Cytosine
Thymine Uracil
 RNA CLASSES AND
FUNCTIONS

MESSENGER RNA (mRNA)

RIBOSOMAL RNA (rRNA)
TRANSFER RNA (tRNA)
 MESSENGER RNA (mRNA)

Linkage between the information from the

DNA and the third step in the central dogma,
that is, translation
An mRNA molecule carries a portion of the
DNA code to other parts of the cell for
processing
mRNA is created during transcription.
 RIBOSOMAL RNA (rRNA)
Important structural and functional part of
ribosomes

Ribosomes are the cellular organelles where

proteins are synthesized

rRNa is the largest component of cellular

RNA,comprising 80-90% of cellular RNA
 TRANSFER RNA (tRNA)
Functions as an adaptor molecule needed in
the reading of the mRNAs by ribosomes, and
there is at least one tRNA of every amino acid
 ISOLATION
AND
PURIFICATION
OF RNA
1960: Kurland and colleagues performed an
isolation procedure using Cesium Chloride
centrifugation technique

1968: Kirby used phenol and chloroform

1979: Chirgwin used guanidium thiocyanate and

beta-mercaptoethanol or ultacentrifugation using
CsCl gradient
1987 Chomczynski and Sacchi combined all
extraction steps in one single procedure
using a reagent composed of guanidium
thiocyanate, phenol,D chloroform

This made made RNA isolation easier and

improved RNA quality and yield
 SPECIMEN
COLLECTION AND
CONSIDERATION

Most critical part in RNA isolation is proper

handling and storage of specimen
Samples should be processed immediately
If immediate processing is not possible,
samples should be stored in liquid nitrogen
or at -80 degree Celsius or in appropriate
preservative agent or in tubes with
stabilizer
In cases where samples will be
collected from sacrificed animals or
from cadavers, the time between
death and sample collection should
be limited and as short as possible.
Other important reminders when performing
RNA isolation are the following:

1. The RNA working area should be different

from the DNA working area (if possible).
2. Gloves should be worn always and
changed frequently to avoid introduction of
"finger RNases."
3. All tubes and bags should be capped and
covered when not in use to avoid
contamination with dust and other
particles.
4. Sterilize area and other materials, such
as pipettors, before and after working. Use
ultraviolet light or spray with Lysol and
alcohol.
 STEPS FOR
RNA
EXTRACTION

The isolation methods for RNA is somewhat

similar with the methods used for DNA
isolation.

Commonly employed methods can be

categorized into organic/inorganic
extraction, adsorption or solid phase
extraction, and the use of isopycnic
gradient centrifugation
There are four major steps in every extraction
procedure, namely:

1. Cell lysis
2. Dehydration and precipitation
3. Separation of cellular proteins and
components
4. Precipitation and dissolution of nucleic acid
Considered as the GOLD STANDARD IN
RNA ISOLATION, the organic/inorganic
extraction method such as phenol-
chloroform technique, starts with sample
lysis using cationic detergent
guanidinium thiocyanate (GTC), followed
by organic extractions and alcohol
precipitation.
As with adsorption methods, the ability of RNA
to form linkage to specific surfaces, such as
beads or tubes, in the presence of chaotropic
salts is utilized. Materials used for beads and
tubes may include, but not limited to, magnet,
polystyrene latex, cellulose matrices, or glass
fibers.
This technique is the most commonly
employed in commercially available RNA
isolation kits.
The third method, which is the
isopycnic gradient method is actually
the first method used in isolating RNA.
This is said to be the method of choice
for isolating RNA free of proteins, DNA
polysaccharides, and other cellular
components.
A common procedure in the
discussed methods of RNA isolation
is the lysis of sample. In cell lysis,
several methods are being used
depending on the source of nucleic
acid
If the source is a:
1. Plant or an animal tissue: samples
are ground in a mortar and pestle
with liquid nitrogen.
2. Samples such as eggs (nematode
metacercariae of some parasites, and
oocysts uses glass-bead grinding
3. When lysing animal cells and zoites
of apicomplexan parasites, such as
sporozoites, merozoites, tahyzoites,
and bradyzoites, as well as of
trypanosomal forms of Trypanosoma
spp. and Leishmania, repetitive
pipetting can be used
Once RNA isolation is finished, the work is not
done yet until the purity, quality, and of RNA is
checked.

This can be done through:

Spectrophotometric assay:
Nucleic acids absorb ultraviolet (UV) light at
260 nm wavelengths
Proteins and phenolic compounds absorb UV
at 280 nm
A satisfactory RNA should have a
ratio of A260/A280 of 1.80 to 2.0
(2.1). Values lower than 1.8
indicates protein or DNA
contamination, or the solution
where RNA is suspended has low
pH.
Absorbance may also be read at 230 nm. At
230 nm, organic compounds, phenol, TRIzol,
and chaotropic salts have strong
absorbances.

A260/A230 should also be above 2.0 (2.0 to

2.2). A low ratio may indicate the
contamination of wash solutions, chaotropic
salts, protein, phenols, and others.
Other methods of checking RNA purity
and concentration are now available but
require different types of equipment such
as Qubit (Thermo Scientific), Experion
(Bio-Rad), and Bioanalyzer 2100 (Agilent),
among others.
In determining the quality of the isolated
RNA, a common method to use is
denaturing gel electrophoresis
 DIAGNOSTIC
SIGNIFICANCE
OF
RNA

Why do we resort to RNA studies if DNA

information is available and is similar across
the cells of an organism?

This is because the genes that are

expressed and the level they are
expressed are different depending on the
physiological state of an individual cell
mRNA expression may show the
overall cellular activity at the
molecular level, and thus,the
molecular regulatory mechanisms
of certain physiologic conditions
like diseasesmaybe explored, and
novel biomarkers may be identified
 RNA
QUANTIFICATION

1. RT-PCR (REVERSE
TRANSCRIPTASE PCR)
2. MICROARRAY
3. RNA SEQUENCING
 PROTEIN
ANALYSIS
Sophia J. Pangilinan, RMT
Complex nitrogen-rich substance
found in cells of all animals and
plants
Not identified until Berzelius
suggested the name "protein" (from
the Greek proteios, which means
"primary")
There are 20 basic amino acids that
make up proteins, and they all have
the same basic structure except for
R-group or side chain they have.
Proteins are the building blocks of
nucleic acid transcription and
translation
In 1942, Martin and Synge developepd
chromatography which is a technique
used to separate proteins
From 1996 to 2003, Mann, Aebersold,
Yates and others developed other
techniques such as mass spectrometr
to identify proteins in complex
mixtures
Proteins are biochemical molecules
made up of covalently linked amino
acid residues and carboxy groups
combined by peptide bonds
Whereas amino acids are the basic
building blocks of proteins.
Amino acids are connected to other
amino acids by an amide bond, which
is referred as a peptide bond
Amino acids combine to form long
linear chains known as polypeptides
STRUCTURE
OF
PROTEINS
Proteins are biochemical molecules made
up of covalently linked amino acid residues
and carboxyl groups combined by peptide
bonds
AMINO ACIDS are the building blocks of
proteins
AMINO ACIDS are connected to other
amino acids by an amide bond, which is
referred to as peptide bond
CLASSIFICATION
OF
AMINO ACIDS
ESSENTIAL AMINO ACIDS: needs to
be included in the diet because the
body cannot synthesize them

NON-ESSENTIAL AMINO ACIDS:

synthesized by the body
ESSENTIAL AMINO ACIDS NON-ESSENTIAL AMINO ACIDS
Histidine Alanine
Arginine
Isoleucine
Asparagine
Leucine Aspartate
Methionine Cystine
Phenylalanine Glutamic acid
Threonine Glycine
Tryptophan Ornithine
Proline
Valine
Serine
Tyrosine

 PROTEIN
CLASSIFICATION
BY CONSTITUTION
SIMPLE PROTEINS: contain only
amino acid residues

CONJUGATED PROTEINS: contain

amino acid residues plus other
organic or inorganic components
referred to as prosthetic group
FUNCTIONS
OF
PROTEINS
IMPORTANT FUNCTION:
ACT AS CATALYSTS
 COMMON
FUNCTIONAL
CLASSES OF
ENZYMES
HYDROLASE Catalyze hydrolytic cleavage reaction
NUCLEASE Breaks down nucleic acids by

hydrolyzing bonds between nucleotides
PROTEASE Breaks down proteins by hydrolyzing

bonds between amino acids
LIGASE Joins two molecules together
ISOMERASE Catalyzes the rearrangeement of bonds

within a single nucleotide
POLYMERASE Catalyzes polymerization reactions
such as the synthesis of DNA and RNA
 Catalyze the addition of phosphate
KINASE
groups to molecules (Protein kinase)

Catalyzes the hydrolytic removal of a

PHOSPHATASE
phosphate group from a molecule

OXIDOREDUCTASE One molecule is oxidized while the

other is reduced;

Enzymes of this type are called oxidase,

reductase and dehydrogenase
ATPase Hydrolyzes ATP
 PARAMETERS/
SUBSTANCES
THAT DENATURE
PROTEINS
 HEAT AND Disrupt hydrogen bonds and ionic
ULTRAVIOLET attractions by making molecules vibrate
LIGHT too violently; produce coagulation

ORGANIC Disrupt hydrogen bonds in proteins and

SOLVENTS probably form new ones with the
(ETHANOL proteins
ANDN
OTHERS
MISCIBLE
WITH WATER)
 STRONG Disrupt hydrogen bonds and ionic
ACIDS OR attactions; prolonged exposure results
BASES in hydrolysis of protein

DETERGENTS Disrupt hydrogen bonds, hydrophobic

interactions and ionic attractions

HEAVY-METAL
Form bonds to thiol groups and
IONS (Hg2+,
precipitate proteins as insoluble heavy-
Ag+ and Pb2+)
metal salts
 PROTEIN
REGULATION
Most of the methods in Proteins Chemistry
involve aqueous media and require
knowledge of pH, pKa and charge on a
peptide at various pH values.

Proteins reversibly change their shape

when ligands bind to their surface.
The regulation of protein activity occur at two
cellular levels.
First, the cell regulates the amount of protein it
contains by controlling the expression of the
gene that encodes the specific protein,
confining sets of enzymes to particular
subcellular compartments
Second, the cell regulates the enzymatic
activities by confining sets of enzymes to
particular subcellular compartments.
PROTEIN
ANALYSIS
 Proteins can be acquired from a broad
range of samples

They can be obtained from:

Cells of tissues of patient
Microorganisms or cell lines derived
from insects, vertebrate animals, or
plants
Regardless of the origin, it is not easy to
obtain a specific protein for several reasons
such as:

(1) No generalized property can be employed

in protein purification.

(2) The quantity of protein of interest tends

to be quite low.
(3) Proteins cannot be amplified.

(4) Problems of contamination are

unavoidable

(5) Proteins are unstable

Using overexpression or enhancement is the
easiest option to handle all the aforementioned
challenges for researchers.
The unique characteristics of each protein,
namely: amino acid composition, sequence,
subunit structure, size, shape, net chage,
isoelectric point, solubility, heat stability,
hydrophobicity, ligand/metal-binding properties
and post-translational modification can be
exploited in the formulation of a strategy for
purification.
 PROTEIN
ISOLATION
Proteins can come from many sources, including
the following:
Natural sources, such as mammalian cell
cultures, tissues, or bodily fluids;
Overexpression in a model system, such as
bacteria, yeast, insect, or mammalian cells;
Monoclonal antibodies from hybridoma
cells;
Plant cells used in agricultural
biotechnology.
Several methods for protein isolation
include:
Mechanical disruption
Liquid homogenization
High-frequency sound waves
Freezing/thaw cycles
Manual grinding
are commonly used for physical lysis cells.
LYSING CELLS
AND
TISSUES
 PHYSICAL
DISRUPTION
PHYSICAL DISRUPTION
MECHANICAL
DISRUPTION
 MECHANICAL DISRUPTION
Mechanical methods depend on the use of
rotating blades to grind and disperse substantial
amounts of complex tissue.
PROCEDURE
The first step in the isolation of proteins is to
disrupt tissues and cells in a regulated fashion
HOMOGENIZATION: will be used to rupture the
plasma membrane of cells to release its
contents.
 MECHANICAL DISRUPTION
This may be accomplished through any of the following
techniques:
1. Use of high-frequency sound to break the cells
2. Use of mild detergent to make holes in the plasma
membrane
3. Force cells through a small hole using high pressure
4. Shear cells between a close-fitting rotating plunger
and the thick walls of a glass vessel
The mixture or solution produced is referred to as a
HOMOGENATE or EXTRACT
 LIQUID HOMOGENIZATION
The most commonly used technique for cell
disruption is the
LIQUID-BASED HOMOGENIZATION.

In this technique, cells are lysed by forcing the

cell or tissue suspension through a narrow
space, which is capable of shearing the cell
membranes.
 FREEZE-THAW
Commonly used to lyse mammalian and
microbial cells which include bacteria,
protozoan and fungi
This is also useful for the disruption of
protozoan cysts, oocysts, and fungal cell wall
such as in cases of giardia, cryptosporidium,
cyclospora, toxoplasma, isospora, and
microsporidia to name a few
 FREEZE-THAW
In this method, the cell suspension is
frozen in a dry ice/ethanol bath or
freezer, and then immediately followed
by thawing at room temperature or 37°C.
This technique causes the swelling of
the cells until they break as ice crystals.
 MORTAR AND PESTLE
Manual grinding is the most basic and common
method used to disrupt cells.
First, the tissue needs to be frozen in liquid
nitrogen and then, crushed using a mortar and
pestle. Because of the tensile strength of the
cellulose and other polysaccharides comprising
the cell wall, for instance of plant cells, this
method is the fastest and most efficient way to
access plant proteins and DNA
 SONICATION
Sonication is another method that can
be used to physically disrupt and break
open cells.
The method uses pulsed, high-frequency
sound waves to agitate and lyse cells,
bacteria, spores, and finely diced tissue.
CHEMICAL AND
ENZYMATIC
METHODS
Microscale cell disruption is often accomplished
through chemical and enzymatic methods or
combinations of the two.
Lysis methods, such as sonication and French
press, are not easily applied to cell pellets
harvested from 5 mL of culture or less, and
excess heat and oxidation are common problems
A significant advancement for simplification of
microscale cell lysis is the development of
detergent-based reagents.
 PROTEIN
SEPARATION
OR
FRACTIONATION
Unlike nucleic acids, proteins do not have a
generalized purification method. The type
isolation and purification technique to be used
actually depends entirely on the physical and
chemical properties of the target protein
Proteins may be purified by liquid
chromatography (LC), and fast protein LC and
high-performance LC can be chosen depending
on whether the goal is for preparatory step only
or for quantitative analysis.
It is possible to use the following techniques as
a single step or as sequentially connected
steps:

Hydrophobic interaction column

chromatography
Size exclusion chromatography
Ion-exchange column chromatography
Affinity chromatography
In general, protein purification necessarily
entails five types of step:

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-
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.
 PROTEIN
SEPARATION BY
CHROMATOGRAPHY
 COLUMN CHROMATOGRAPHY
Column chromatography is a primary protein
purification method used in most
laboratories. A combination of proteins in
solution is added to the surface of a
cylindrical column loaded with a porous solid
matrix submerged in a solvent. After that, a
large amount of solvent is pumped through
the column.
 COLUMN CHROMATOGRAPHY
Due to their association with the matrix,
separated proteins are deluded to distinct
levels, and they can be obtained individually
as they flow from the bottom. Proteins can be
separated depending on their charge,
hydrophobicity, size, or ability to attach to
specific chemical components depending on
matrix selection
ION-EXCHANGE CHROMATOGRAPHY
Columns of ion-exchange are filled with tiny
beads holding either positive or negative
charges that retard proteins of the opposite
charge.
The interaction of protein and matrix relies on
the pH and ionic strength of the fluid moving
down the column.
GEL FILTRATION CHROMATOGRAPHY
Columns of gel-filtration separate proteins by
size. The matrix is made up of small porous
beads. Protein molecules tiny enough to reach
the bead holes are postponed and move
through the column more slowly. First,
proteins that cannot enter the beads are
wiped out of the column. Such columns also
enable protein size estimation.
 AFFINITY CHROMATOGRAPHY
Affinity columns contained in a matrix
covalently pair with a molecule that
interacts specifically with the protein of
interest. Proteins that attach specifically to
such a column can eventually be produced
through a pH shift or concentrated salt
solutions, and these are extremely purified.
PROTEIN ANALYSIS
(QUANTITATIVE)
 ENZYME-LINKED
IMMUNOSORBENT
ASSAY
(ELISA)
Enzyme-linked immunosorbent assay (ELISA)
is a method of target antigen (or antibody)
capture in samples using a specific antibody
(or antigen) and target molecule
detection/quantitation using an enzyme
reaction with its substrate
ELISA is an enzyme-amplified reaction for
antigens present in trace amounts in bodily
fluids.
The ELISA technique is a sensitive assay--
both the presence of the target protein and
quantitative information about it can be
obtained without purification.

In ELISA, various combinations of antigen-

antibody, including an enzyme-labeled
antigen or antibody, are used, and the
enzyme activity is measured colorimetrically.
 DIRECT ELISA
A target protein (or a target antibody) is
immobilized on the surface of microplate
wells and incubated with an enzyme-
labeled antibody specific to the target
protein. Substrate is added, and reaction
is observed and measured using a
microplate reader.
 INDIRECT ELISA
On the surface of microplate wells, a target
protein is immobilized and incubated with a
primary antibody specific to the target protein.
This is followed by the addition of an enzyme-
conjugated secondary antibody that will bind to
the primary antibody. The activity of the well-
bound microplate enzyme is measured after
adding the substrate and terminating the
reaction.
 SANDWICH ELISA
A target protein (or a target antibody) is
immobilized on the surface of microplate wells and
incubated with an enzyme-labeled antibody to the
target protein (or a specific antigen to the target
antibody). After washing, the activity of the
microplate well-bound enzyme is measured. The
immobilized (orange) antibody and the enzyme-
labeled (green) antibody must recognize various
epitopes of the target protein.
 COMPETITIVE ELISA
An antibody specific to a target protein is
immobilized on the surface of microplate wells
and incubated with samples containing the target
protein and a known amount of enzyme-labeled
target protein. After the reaction, the activity of
the well-bounded microplate enzyme is measured
 COMPETITIVE ELISA
As this is competitive ELISA, color reaction is
inversely proportional to the concentration of the
target protein
When the antigen level in the sample is high, the
level of antibody-bound enzyme-labeled antigen
is lower and the color is lighter.
Conversely, when it is low, the level of antibody-
bound enzyme-labled antigen is higher, and the
color is darker