KATHMANDU UNIVERSITY

Dhulikhel, Kavreplanchowk
Nepal

Assignment: 1
Subject: CHEG 213

Submitted by: Submitted to:

Ramesh Pokhrel Dr. Bishnu Prasad Pandey
2nd year, 2nd sem Department of
Group:CheE Chemical Science &Engineering
Roll No:22

Date: -July 30th, 2018

1) The difference between Anabolic and Catabolic Reactions is given below:
Anabolism Catabolism
Anabolism is the metabolic process Catabolism is the metabolic process which
where simple substances are synthesized into breaks down large molecules into smaller
complex molecules. molecules.
Anabolism is the constructive phase of Catabolism is the destructive phase of
metabolism. metabolism.
Anabolism requires ATP energy. Catabolism releases ATP energy
Anabolism is an endergonic reaction. Catabolism is an exergonic reaction.
Estrogen, testosterone, growth hormone, Adrenaline, cortisol, glucagon, cytokines,
insulin, etc. are involved in anabolism. etc. are involved in catabolism.
Anabolism is anaerobic; it does not utilize Catabolism is aerobic; it utilizes oxygen.
oxygen.
Kinetic energy is converted into potential Potential energy is converted into kinetic
energy during anabolism. energy during catabolism.

The Embden-Meyerhof-Parnas (EMP) pathway allows the metabolic use of glucose to generate

ATP, NADH, and several biosynthetic precursors such as 3-phosphoglycerate or pyruvate. The
EMP pathway can occur both anaerobically (leading to one or several fermentation pathways)
and aerobically through the conversion of pyruvate to acetyl CoA and the connection with the
tricarboxylic acids (TCA) cycle. The classical version of the EMP pathway is present in bacteria
and eukaryotes whereas several modified versions are present in anaerobic archaea. The second
half of the pathway is almost universal, and thus, it could represent the oldest part of the pathway,
related to a primordial origin of gluconeogenesis.
The EMP steps involve isomerization, ring splitting, or transfer of a small group such as hydrogen
or phosphate. Two moles of pyruvate are produced per mole of glucose passing through the
pathway.
The overall stoichiometry of the EMP pathway is

Stored chemical energy and reducing power result from overall pathway. Energy storage
accomplished by this or other substrate rearrangement pathways is called substrate-level
phosphorylation.

In muscle cell and lactic acid bacteria, the reactions of the EMP are followed by single step

This
overall reaction sequence from glucose to lactic acid is called glycolysis. It is interesting to
compare the free energy change for glycolysis

With corresponding quantity for the glucose breakdown alone

A total free-energy of 14.6 kcal or 7.3 kcal for each mole of ATP generated has been conserved
by the pathway as high energy phosphate compounds.
2) Cellular respiration is a set of metabolic reactions and processes that take place in
the cells of organisms to convert biochemical energy from nutrients into adenosine
triphosphate (ATP), and then release waste products. The reactions involved in respiration
are catabolic reactions, which break large molecules into smaller ones, releasing energy in the
process, as weak so-called "high-energy" bonds are replaced by stronger bonds in the products.
Tricarboxylic acid (TCA) cycle, also called Krebs cycle or citric acid cycle is the central
metabolic hub of the cell.
The reactions of TCA cycle are described below:

 Citrate synthase: Formation of Citroyl CoA intermediate.

 Binding of Oxaloacetate to the enzyme results in conformational change which facilitates the
binding of the next substrate, the acetyl Coenzyme A. There is a further conformational change
which leads to formation of products. This mechanism of reaction is referred as induced fit
model.

 Aconitase: This enzyme catalyses the isomerization reaction by removing and then adding back
the water (H and OH) to cis-aconitat in at different positions. In this way produced Isocitrate
consume rapidly in next step.
 Isocitrate dehydrogenase: There are two isoforms of this enzymes, one used NAD+ and other
uses NADP+ as electon acceptor. The enzyme isocitrate dehydrogenase convert isocitrate to
alpha-ketoglutarate

 alpha-

ketoglutarate dehydrogenase: This is a complex of different enzymatic activities similar to the

pyruvate dehydrogenase complex. The alph-ketoglutarate is converted to Succinyl-CoA by
enzyme called alpha-ketoglutarate dehydrogenase

 Succinyl CoA synthatase: Succinyl CoA , likely Acetyl CoA has a thioester bond with very
negative free energy of hydrolysis
 Succinate Dehydrogenase: Oxidation of succinate to fumarate. This is the only citric acid cycle
enzyme that is tightly bound to the inner mitrochondrial membrane. It is an FAD dependent
enzymes

 Fumarase: Hydration of fumarate to malate: It is a highly sterospecific enzyme.

 L-malate dehydrogenase: oxidation of malate to oxaloacetate: It is an NAD+ dependent

enzyme. Reaction is pulled in forward direction by the next reaction (citrate synthase reaction)
as the oxaloacetate is depleted at a very fast rate
 #3) The microbial growth curve is given below:

1) Lag phase: In this phase, bacteria adapt themselves to growth conditions. It is the period where
the individual bacteria are maturing and not yet able to divide. During the lag phase of the
bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.
2) The log phase (sometimes called the logarithmic phase or the exponential phase) is a period
characterized by cell doubling. The number of new bacteria appearing per unit time is
proportional to the present population. If growth is not limited, doubling will continue at a
constant rate so both the number of cells and the rate of population increase doubles with each
consecutive time period.
3) The stationary phase is often due to a growth-limiting factor such as the depletion of an essential
nutrient, and/or the formation of an inhibitory product such as an organic acid. Stationary phase
results from a situation in which growth rate and death rate are equal. The number of new cells
created is limited by the growth factor and as a result the rate of cell growth matches the rate of
cell death.
4) At death phase (decline phase), bacteria die. This could be caused by lack of nutrients,
environmental temperature above or below the tolerance band for the species, or other injurious
conditions.

Monod Chemostat Model:

A functional relationship between the specific growth rate µ and essential compound’s
concentration was proposed by Monod in 1942.

Here µmax is the maximum growth rate achieved and Ks is a saturation constant, when s>>Ks
and the concentrations of all other essential nutrients are unchanged.
 Ks is that value of the limiting nutrient concentration at which the specific growth rate is half its
maximum value.
The steady-state mass balance on substrate is then

Putting the value of µ we get the corresponding cell mass balance is

These two equation are called Monod chemostat model equation.

5) Difference between exon and introns is given below:

Introns Exons
Introns are DNA segments which do not Exons are the DNA segments which encode
encode any amino acid sequence in the a part of an amino acid sequence of a
coding region. complete protein.
Introns belong to the non-coding DNA. Exons belong to the coding DNA.
Introns are considered as the bases located Exons are the bases which encode an
between two exons. amino acid sequence of a protein.
Introns are only found in eukaryotes. Exons are found in both prokaryotes and
eukaryotes.
The sequences in the introns are less The sequences in the exons are highly
conserved compared to exons. conserved.
Introns are found in DNA and mRNA Exons are found in both DNA and mRNA.
primary transcript.
 DNA replication:

 DNA contains the information required for proper development and function of the living cell, it
is essential that there be an almost fool proof mechanism for copying DNA.
 When a cell produces, each of the offspring must receive a complete set of genetic data in the
form of DNA
 After the original strands unwind, new complementary chain are assembled, so that ultimately
two intact DNA molecule result.
 DNA polymerase conducts synthesis of daughter strands in the 5' 3' direction. This proceeds
relatively smoothly on the parental strand running 3' 5'. On the other parental strand, DNA
polymerase adds fragments of the daughter strand in the 5' 3' direction which are then covalently
coupled by the enzyme DNA ligase.
 This achieves overall daughter strand synthesis in the 3' 5' direction
 Each strand of the parent DNA is used as a template to make the new daughter strand
 DNA replication makes 2 new complete double helices each with 1 old and 1 new strand.
 An enzyme that catalyses the addition of a nucleotide to the growing DNA chain
 Nucleotide enters as a nucleotide tri-PO4
 3’–OH of sugar attacks first phosphate of tri-PO4bond on the 5’ C of the new nucleotide releasing
pyrophosphate (PPi) + energy

MUTATION:
Mutation, an alteration in the genetic material (the genome) of a cell of a living organism or of
a virus that is more or less permanent and that can be transmitted to the cell’s or the virus’s
descendants. (The genomes of organisms are all composed of DNA, whereas viral genomes can
be of DNA or RNA; see heredity: The physical basis of heredity.) Mutation in the DNA of a
body cell of a multicellular organism (somatic mutation) may be transmitted to descendant
cells by DNA replication and hence result in a sector or patch of cells having abnormal function,
an example being cancer. Mutations in egg or sperm cells (germinal mutations) may result in
an individual offspring all of whose cells carry the mutation, which often confers some serious
malfunction, as in the case of a human genetic disease such as cystic fibrosis. Mutations result
either from accidents during the normal chemical transactions of DNA, often during replication,
or from exposure to high-energy electromagnetic radiation (e.g., ultraviolet light or X-rays) or
particle radiation or to highly reactive chemicals in the environment. Because mutations are
random changes, they are expected to be mostly deleterious, but some may be beneficial in
certain environments. In general, mutation is the main source of genetic variation, which is the
raw material for evolution by natural selection.

5) Explain the major steps involve in gene cloning. How recombinant protein undergo transcription
and translation.
The major steps of gene cloning are given below:
 Cutting the DNA to be cloned from the chromosomal using sequence specific Restriction
Endonuclease.
 Selecting a cloning vector (a small molecule capable of self-replicating inside host cells), and
cutting the cloning vector with the same restriction endonuclease producing the cohesive ends.
 Incubating the vector and subject DNA together to anneal and then joining them using DNA
ligase. The resultant DNA is called recombinant DNA.
 Transferring the recombinant DNA to an appropriate host such as bacteria, virus or yeast which
will provide necessary biomachinary for DNA replication.
 Identifying the host cells that contain the recombinant DNA.

6) Briefly explain the Watson’s and Crick’s model of DNA.

1. Structurally, DNA is made up of nucleotides.

2. A nucleotide is made up of a sugar, a phosphate and a base.
3. Nucleotides hook together to make the sugar-phosphate backbone of every strand of DNA.
4. Bases pair up with opposite, matching bases on the other strand of DNA.
5. There are four kinds of base pairs made up of adenine (A), thyamine (T), guanine (G),
cytosine (C). A always pairs with T. G always pairs with C.
6. The two strands of DNA are linked together by weak hydrogen bonds and stacked like a
ladder who’s sides spiral around each other into their famous double-helix shape.

8. Differentiate between primary and secondary structure of protein.

Primary structure Secondary Structure
Amino acids are joined together by peptide It arises due to the regular folding of the
linkage. poly peptide chain due to intermolecular H
bonding between c=o and NH2 groups.
 The sequence in which the amino acids are The peptide chain can be coiled to form a
arranged is called primary structure. helix structure or they can be stretched out to
form β structure.

9. Shed light on the basic thermodynamics principles of biochemical reaction.

To knowing the certain reaction in the cell that will run forward or backword, we will use a
number of approximation since full analysis of metabolic network is not practical. First let us
consider the free-energy change of a chemical reaction.

αA βB  γC δD
This equation can be written in the form

ΔG ΔG0RTln cℽ d δ
aα bβ

Where, ΔG0' is the standard free energy change

In a closed system, the reaction will proceed left to right if and only if ΔG' is negative.
Accordingly, ΔG' is zero at equilibrium give the following relationship:

ΔG0-RTlnKeq
Where Keq=ceq deq aeq beq

CHO
CH2 OH 0
CHOH G ’= -1830 cal/mol
C O
CH2 O P
CH2 O P
Glyceraldehyde
3-phosphate Dihydroxyacetone-P

Where P denotes phosphate. Because of the negative free energy change, equilibrium favours
the dihydroxyacetone by a 22:1 ratio.
Many biological reaction and energy conversion process involve oxidation-reduction reaction
such as

Aox + Bred = Ared + Box

This type of reaction is described using the standard potential change.
 Where is the standard half-cell potential for the half reaction

As a reference point for half-cell potential value, the hydrogen half-cell (at pH=0) is assigned a
value of zero:

The free energy change and corresponding potential changes are related by

Where n is the number of electrons transferred and F is equal to 23.062 kcal/V mol.

10. Briefly explain the pentose phosphate pathway and also calculate the energetics of PPP.

The pathway begins with the glycolytic intermediate glucose 6-P.It reconnects with glycolysis
because two of the end products of the pentose pathway are glyceraldehyde 3-P and fructose 6-
P; two intermediates further down in the glycolytic pathway.It is for this reason that the pentose
pathway is often referred to as a shunt.
Also known as: Pentose shunt or Hexose monophosphate shunt or Phosphogluconate pathway
The pathway yields reducing potential in the form of NADPH to be used in anabolic reactions
requiring electrons.
 The pathway yields ribose 5-phosphate.
 Nucleotide biosynthesis leading to:
 DNA
 RNA
 Various cofactors (CoA, FAD, SAM, NAD+/NADP+).
Pentose phosphate cycle or pathway (also called the hexose monophosphate or shunt) begins by
oxidizing glucose phosphate

The major function of the pentose phosphate pathways is supplying the cell with NADPH
which in turn carries electrons to biosynthesis reaction
The total reaction scheme is quite complicated, but its overall result is indicated by the following
summary of pentose phosphate pathway stoichiometry:

The net effect is complete oxidation of one mole of glucose 6-phosphate liberating CO2and
transferring all of the electrons (H) to NADP.This overall reaction consumes ATP. In order to
provide ATP to the cell, an incomplete pentose phosphate cycle may occur with overall
stoichiometry

Pentose phosphate pathway is advantageous in the sense of including ribose 5-phosphate and
erythrose 4-phosphate, important precursors for purine and pyrimidine biosynthesis, among its
intermediates. These components are absent from the EMP pathways
As a consequence, microorganisms such as lactobacilli which posses only EMP pathway require
a complex medium including pentose, purines and pyrimidines when grown in anerobic
environment.
E. coli, on the other hand is able to grow anaerobically in simpler media using the EMP and
pentose phosphate pathways simultaneously ( for example, in the ratio of 75% EMP: 25%
pentose phosphate pathways under certain culture conditions)
11. What is the role of promotor in transcription? Briefly explain the genetic code.
A promoter region is a particular DNA sequence within a DNA strand that acts as a signal for
where RNA transcription should begin. It includes the binding site for RNA polymerase, the
transcription start site, and sites where proteins that are responsible for regulating gene
expression can bind. A transcription stop site occurring further on the DNA strand signals the
end of transcription.
As you may recall, DNA is made up of nucleotides. As such, a promoter is a sequence of
nucleotides. A nucleotide is a molecule that is made up of a nitrogenous base, a pentose (5
carbon) sugar, and a phosphate group. The sequence of these nucleotides, differentiated by their
nitrogenous bases (adenine, thymine, guanine and cytosine), will identify a promoter region.

GENETIC CODE:
The genetic code = the sequence of bases found along the mRNA molecule. There are only four
letters to this code (A, G, C and U)
The code needs to be complex enough to represent 20 different amino acids used to build
proteins.
If one base represented one amino acid this would only be able to produce 4 different
combinations. (A, C, G and U)
If pairs of bases represented each amino acid this would only be able to produce… 4 x 4 = 16
combinations. (AA, AC, AG, AU, CA, CC, CG, CU etc.)
If triplets of bases represented each amino acid, this would be able to produce… 4 x 4 x 4 = 64
combinations. This is enough combinations to code for the 20 amino acids. The genetic code is
made of triplets of bases called codons.
12. Sidelight on posttranslational modification of protein.
Post-translation modification refers to modification of protein during or after protein
biosynthesis (covalent or enzymatic). Protein are synthesized by ribosome translation mRNA
into polypeptide chains, which may then undergo post-translation modification to form the
mature protein products. The polypeptide produced by mRNA translation is often not the final
biologically active form of the molecules. The N-terminal methionine is sometimes removed.
Two cysteine residues may be oxidized to give a disulphide bond which can be a significant
factor in protein tertiary structure. Other types of polypeptide chemical modification include
hydroxylation, phosphorylation and acetylation. Some polypeptide have at their N-terminus a
short (15-20 residues) sequence of hydrophobic residues. These signal sequence are important
components in protein transport across cell membrane. Secreted proteins generally contain such
signal sequence, and denoted by prefix “pre” to the name of a protein to indicate the form
which includes the signal sequences. Eg. Prelysozymes. Preprotein has crossed the membrane,
its signal sequence is removed to give the mature protein (lysozyme in above example)

14. Write a simplified classification of microorganism. Briefly explain the elemental composition
of bacteria.
 15. What are the basic features of amino acids? Draw the structure of any two polar and non-
polar amino acids.
The basic features of amino acids are given below:
 Amino acids are the building blocks of proteins
 There are twenty amino acids present in all living organism, which is clear manifestation of the
biological unity in all living organism
 These twenty amino acids have different structural configurations and are represented by three
latter symbol.
 They are also represented by single alphabets which help in writing the amino acid sequence in
the protein.
 Some protein contain a large combination of several amino acids
 Two polar and non-polar amino acids are given below:

16. Distinguish between entrapment and surface immobilization techniques used for enzyme
immobilization.
Entrapment Surface immobilization
Entrapment is the physical enclosure of Immobilization is defined as the
enzyme in a small space. imprisonment of cell or enzyme in a distinct
support or matrix.

Matrix entrapment and membrane Two major types of immobilization of

entrapment including microencapsulation are enzyme on the surface of support materials
the two major methods for entrapment are adsorption and covalent binding

17. What is gene expression? Briefly explain the process of transcription and translation.
Gene expression is the process by which genetic instructions are used to synthesize gene
products. These products are usually proteins, which go on to perform essential functions as
enzymes, hormones and receptors, for example. Genes that do not code for proteins such as
ribosomal RNA or transfer RNA code for functional RNA products.
 Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make
an RNA molecule.
 RNA polymerase is the main transcription enzyme.
 Transcription begins when RNA polymerase binds to a promoter sequence near the beginning
of a gene (directly or through helper proteins).
 RNA polymerase uses one of the DNA strands (the template strand) as a template to make a
new, complementary RNA molecule.
 Transcription ends in a process called termination. Termination depends on sequences in the
RNA, which signal that the transcript is finished.
Transcription is the process by which the information in a strand of DNA is copied into a new
molecule of messenger RNA (mRNA). DNA safely and stably stores genetic material in the
nuclei of cells as a reference, or template. Meanwhile, mRNA is comparable to a copy from a
reference book because it carries the same information as DNA but is not used for long-term
storage and can freely exit the nucleus. Although the mRNA contains the same information, it is
not an identical copy of the DNA segment, because its sequence is complementary to the DNA
template.
Transcription is carried out by an enzyme called RNA polymerase and a number of accessory
proteins called transcription factors. Transcription factors can bind to specific DNA sequences
called enhancer and promoter sequences in order to recruit RNA polymerase to an appropriate
transcription site. Together, the transcription factors and RNA polymerase form a complex called
the transcription initiation complex. This complex initiates transcription, and the RNA
polymerase begins mRNA synthesis by matching complementary bases to the original DNA
strand. The mRNA molecule is elongated and, once the strand is completely synthesized,
transcription is terminated. The newly formed mRNA copies of the gene then serve as blueprints
for protein synthesis during the process of translation.
Translation:
In molecular biology and genetics, translation is the process in which ribosomes in the
cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA in the
cell's nucleus. The entire process is called gene expression.
In translation, messenger RNA (mRNA) is decoded in a ribosome to produce a specific amino
acid chain, or polypeptide. The polypeptide later folds into an active protein and performs its
functions in the cell. The ribosome facilitates decoding by inducing the binding
of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific
amino acids that are chained together into a polypeptide as the mRNA passes through and is
"read" by the ribosome.
Translation proceeds in three phases:
Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at
the start codon.
Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon.
The ribosome then moves (translocates) to the next mRNA codon to continue the process,
creating an amino acid chain.
Termination: When a stop codon is reached, the ribosome releases the polypeptide.
 18. Draw the structure of the predominant Purines and pyrimidines bases found in DNA, name
them.

19. What are lipids? How lipid form monolayer and bilayer.
Lipids are an important component of living cells. Together with carbohydrates and proteins,
lipids are the main constituents of plant and animal cells.
Cholesterol and triglycerides are lipids. Lipids are easily stored in the body. They serve as a
source of fuel and are an important constituent of the structure of cells.
Lipids include fatty acids, neutral fats, waxes and steroids (like cortisone). Compound lipids
(lipids complexed with another type of chemical compound) comprise the lipoproteins,
glycolipids and phospholipids.

A monolayer at air-water interface

Lipid molecules form a monolayer at an air- water interface. Hydrophilic head groups point
into the water phase, while hydrophobic chains point into the air. Along the z- direction, which
is perpendicular to the interface, the monolayer also has an ordered structure of several layers
 On top of the water sub phase there is a layer of hydrophilic head group’s layer
 On top of the head group layer there is a layer of hydrophobic chain layer
 On top of the hydrophobic chain layer there is the air super phase

Bilayers of lipids
 Besides the monolayer structure, lipids can also form bilayer structure in hydrophilic
surrounding like in water. A bilayer can be considered as two monolayer sheets that one is on
top of the other. Bilayer in water has the " sandwich structure" that head group layers face to
each side while two chain layer contact with each other in between two head group layer.
Along the z-direction a bilayer has a particular layers order as following:
 on top of the hydrophilic sub phase there is the firts head group layer from the first lipid
monomer
 on top of the first head group layer there is the first chain layer from the second lipid monolayer
 on top of the first chain layer there is the second chain layer from the second lipid molecules
 on top of the second chain layer there is the second head group layer from the second lipid
monolayer
 on top of the second head group layer there is the hydrophilic super phase

21. Distinguish between protein and peptide. How competitive inhibitors affects the rate of
enzyme reaction.
A competitive inhibitor (I) competes with the substrate for binding to the active site of an enzyme.
While the inhibitor occupies the active site, it prevents the binding of the substrate to the enzyme
and blocks the reaction. Many competitive inhibitors are structurally similar to the substrate and
combine with the enzyme to form an EI complex, but without leading to catalysis. Competitive
inhibition can be analysed quantitatively by steady-state kinetics. In the presence of a competitive
inhibitor, the MM equation becomes

The experimentally determined variable αKm, the Km observed

in the presence of the competitive inhibitor, is often called the
“apparent” Km.
Protein Peptide
A protein is a complex structure made out of Peptides are short chains of amino acid
a large number of amino acids. monomers linked by peptide (amide) bonds.
Proteins are made from polypeptides. Peptides are formed from amino acids.
 The molecular weight of a protein is higher
than a polypeptide.
Proteins are composed of several types of
bonds such as peptide bonds, disulfide
bonds, ionic bonds and Van Der Waal
attractions.
The molecular weight of a peptide is lower
than that of a protein.
Polypeptides are composed of peptide
bonds.

24. What is recombinant DNA technology? Explain in detail the polymerase chain reaction.
Recombinant DNA technology, joining together of DNA molecules from two
different species that are inserted into a host organism to produce new genetic combinations that
are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is
the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate
genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding
a specific gene within this DNA sample can be compared to finding a needle in a haystack.
Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA.
Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant
DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling
researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific
ways, and reinsert the modified sequence into a living organism.
Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single
copy or a few copies of a segment of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence.
Reaction involve:
1. DNA template
2. Primers
3. Enzyme (DNA polymerase)
4. dNTPs
5. Mg2+
6. buffers

The PCR reaction requires the following components:

DNA template-the sample DNA that contains the target sequence. At the beginning of the
reaction, high temperature is applied to the original double-stranded DNA molecule to separate
the strands from each other.
DNA polymerase-a type of enzyme that synthesizes new strands of DNA complementary to the
target sequence. The first and most commonly used of these enzymes isTaqDNA
polymerase(fromThermis aquaticus), whereasPfuDNA polymerase (fromPyrococcus furiosus)
is used widely because of its higher fidelity when copying DNA. Although these enzymes are
subtly different, they both have two capabilities that make them suitable for PCR: 1) they can
generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant.
Primers-short pieces of single-stranded DNA that are complementary to the target sequence.
The polymerase begins synthesizing new DNA from the end of the primer.
Nucleotides (dNTPs or deoxynucleotide triphosphates)-single units of the bases A, T, G, and C,
which are essentially "building blocks" for new DNA strands.
PCR reaction steps:
Comprised of 3 steps:
-Denaturation of DNA at 950C
-Primer hybridization ( annealing) at 40-500C
-DNA synthesis ( Primer extension) at 720C

25. Differentiate between prokaryotes and eukaryotes.

 Prokaryotes
Flagellum is single stranded without
differentiation of sheath and axoneme.
Membrane bound organelles are absent.
Ribosomes is of 70 S type (50 S +30 S).
Cell division is only amitosis.
Gametes formation is not possible.
Ratio of DNA and RNA is about 1:2.
Naked dsDNA (no histone protein) or
genophore.
Absence of transcription and translation.
The messenger RNA is polycistronic.
Eukaryotes
The flagellum is eleven (9+2) stranded with
differentiation of sheath and axoneme.
Present
Ribosomes is of 70 S type (50 S and 30 S)
and 80 S type (60 S +40 S).
Amitosis, mitosis, and meiosis
Possible and always haploid.
Ratio of DNA and RNA is about 1:1.
dsDNA with histone protein and forms
eukaryotic chromosome.
Both occurs in nucleus, cytoplasm, and
organelles.
Monocistronic.

Southern blotting:
Screening for specific nucleotide sequences in the clone may be accomplished by several
different procedures which are all based on hybridization of denatured (i.e. Single stranded)
DNA from the clone with a nucleic acid probe, typically labelled with
32Ptomakeitradioactive.In southern blotting method, the DNA in the gel is denatured with alkali,
and the gel is covered with cellulose nitrate filter paper. Buffer solution drawn from moist filter
paper below this sandwich to dry filter paper above it carries DNA from the gel to the cellulose
nitrate paper where it binds. This bound denatured DNA is then exposed to probe and then to x-
ray film as before to identify gel bands containing nucleotide sequence complementary to that of
the probe.

Hemicellulose and lignin:

Hemicellulose is a smaller branched carbohydrate which can be made of different
monosaccharides, cellulose is made of very long unbranched fibrils composed exclusively of
glucose, held together by hydrogen bonding.
Hemicellulose
Second most abundant biopolymer on earth.
Hemicellulose: Heteropolymer of pentoses (xylose and arabinose) and hexoses (glucose,
galactose, mannose) and sugar acids .Hemicelluloses are made of xylan (xylose, arabinose and
glucuronic acid) backbone. Xylan backbone is highly substituted by side chains consisting of
xylose, arabinose and galucuranic acids
Heteroxylans are highly cross linked with diferulic bridges (with other xylans), isodityrosine
bridges (structural proteins). Hemicellulose can be extracted by alkali or hot water and
hydrolysed by dil. H2SO4.

Lignin
A racemic, heteropolymer consisting of three hydroxycinnamyl alcohol monomers differing in
their degree of methoxylation: p-coumaryl, coniferyl and sinapyl alcohols
 Vector and restriction enzyme:
Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a DNA
molecule at a particular place. They are essential tools for recombinant DNA technology. The
enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six
nucleotides. Substrate –DNA -recognizes one particular nucleotide sequence in DNA and cuts
the DNA molecule (breaks down the bond between two nucleotides).
Nomenclature
EcoRI
E = Escherichia genus name
co = colispecies name
R = strain RY12strain or serotype
I = Roman numeral one = first enzyme

Vector are DNA molecule which provide propagation of a DNA fragment in a growing cell
population. A useful vector for cloning should have the following properties
 ability to replicate in the host cell
 ability to accommodate foreign DNA of various sizes without damage to replication functions
 easy insertion in host cell after invitro DNA manipulation
 contains a selectoin marker to facilitate rapid, positive selection of cells which contain vector
 contain only one target site for one or more different restriction endonuclease
Two different classes of vectors -plasmid and bacteriophages have been developed for cloning
DNA in E. coli.