STUDY MATERIAL FOR

B.G 5th Semester

SUBJECT: BIOTECHNOLOGY
GENETIC ENGINEERING
GENETIC ENGINEERING

Genetic engineering primarily involves the manipulation of genetic material (DNA) to achieve
the desired goal in a pre-determined way. Some other terms are also in common use to describe
genetic engineering.
 Gene manipulation
 Recombinant DNA (rDNA) technology
 Gene cloning (molecular cloning)
 Genetic modifications
 New genetics
A clone is an identical copy. This term originally applied to cells of a single type, isolated and
allowed to reproduce to create a population of identical cells. DNA cloning involves separating a
specific gene or DNA segment from a larger chromosome, attaching it to a small molecule of
carrier DNA, and then replicating this modified DNA thousands or millions of times through
both an increase in cell number and the creation of multiple copies of the cloned DNA in each
cell. The result is selective amplification of a particular gene or DNA segment.

MOLECULAR CLONING
The basic procedure of molecular cloning involves a series of steps.
1. Cutting DNA at precise locations. Sequence-specific endonucleases (restriction
endonucleases) provide the necessary molecular scissors.
2. Selecting a small molecule of DNA capable of self-replication. These DNAs are called
cloning vectors (a vector is a delivery agent). They are typically plasmids or viral DNAs.
3. Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning vector
and DNA to be cloned. Composite DNA molecules comprising covalently linked
segments from two or more sources are called recombinant DNAs.
4. Moving recombinant DNA from the test tube to a host cell that will provide the enzymatic
machinery for DNA replication.
5. Selecting or identifying host cells that contain recombinant DNA.

The methods used to accomplish these and related tasks are collectively referred to as
recombinant DNA technology or, more informally, genetic engineering.

CLONING VECTORS
Cloning vectors are carrier DNA molecules. Four important features of all cloning vectors are
that they: (i) can independently replicate themselves and the foreign DNA segments they carry;
(ii) contain a number of unique restriction endonuclease cleavage sites that are present only once
in the vector; (iii) carry a selectable marker (usually in the form of antibiotic resistance genes or
genes for enzymes missing in the host cell) to distinguish host cells that carry vectors from host
cells that do not contain a vector; and (iv) are relatively easy to recover from the host cell. There
are many possible choices of vector depending on the purpose of cloning. The greatest variety of
cloning vectors has been developed for use in the bacterial host E. coli. Thus, the first practical
skill generally required by a molecular biologist is the ability to grow pure cultures of bacteria.

Choice of vector is dependent on insert size and application

The classic cloning vectors are plasmids, phages, and cosmids, which are limited to the size
insert they can accommodate, taking up to 10, 20, and 45 kb, respectively (Table below).
A cosmid is a plasmid carrying a phage  cos site, allowing it to be packaged into a phage head.
Cosmids infect a host bacterium as do phages, but replicate like plasmids and the host cells are
not lysed. Mammalian genes are often greater than 100 kb in size, so originally there were
limitations in cloning complete gene sequences. Vectors engineered more recently have
circumvented this problem by mimicking the properties of host cell chromosomes. This new
generation of artificial chromosome vectors includes bacterial artificial chromosomes (BACs),
yeast artificial chromosomes (YACs), and mammalian artificial chromosomes (MACs).

PLASMIDS AS CLONING VECTORS

Plasmids are circular DNA molecules that replicate separately from the host chromosome.
Naturally occurring bacterial plasmids range in size from 5,000 to 400,000 bp. They can be
introduced into bacterial cells by a process called transformation. The cells (generally E. For
reasons not well understood, some of the cells treated in this way take up the plasmid DNA coli)
and plasmid DNA are incubated together at 0 °C in a calcium chloride solution, then subjected to
a shock by rapidly shifting the temperature to 37 to 43 °C.. Some species of bacteria are naturally
competent for DNA uptake and do not require the calcium chloride treatment. In an alternative
method, cells incubated with the plasmid DNA are subjected to a high-voltage pulse. This
approach, called electroporation, transiently renders the bacterial membrane permeable to large
molecules.
Regardless of the approach, few cells actually take up the plasmid DNA, so a method is needed
to select those that do. The usual strategy is to use a plasmid that includes a gene that the host
cell requires for growth under specific conditions, such as a gene that confers resistance to an
antibiotic. Only cells transformed by the recombinant plasmid can grow in the presence of that
antibiotic, making any cell that contains the plasmid “selectable” under those growth conditions.
Such a gene is called a selectable marker.
Investigators have developed many different plasmid vectors suitable for cloning by modifying
naturally occurring plasmids. The E. coli plasmid pBR322 offers a good example of the features
useful in a cloning vector (Fig. 9-4 ):
1. pBR322 has an origin of replication, ori, a sequence where replication is initiated by cellular
enzymes (Chapter 25). This sequence is required to propagate the plasmid and maintain it at a
level of 10 to 20 copies per cell.
2. The plasmid contains two genes that confer resistance to different antibiotics (tetR, ampR),
allowing the identification of cells that contain the intact plasmid or a recombinant version of the
plasmid (Fig. 9–5).
3. Several unique recognition sequences in pBR322 (PstI, EcoRI, BamHI, SalI, PvuII) are targets
for different restriction endonucleases, providing sites where the plasmid can later be cut to
insert foreign DNA.
4. The small size of the plasmid (4,361 bp) facilitates its entry into cells and the biochemical
manipulation of the DNA.
BACTERIOPHAGES AS CLONING VECTORS

Bacteriophages are the viruses that infect bacterial cells after infecting their genetic material
(DNA or RNA) and kill them. The viral DNA replicates and expresses inside the bacterial cells
and produces a number of phage particles released after bursting the bacterial cells. This is called
lytic cycle of bacteriophage. The released phage re-infects the live cells. The ability of
transforming the viral DNA from phage capsid specific bacterial cell gave insight to the
scientists to exploit bacteriophages and design them as cloning vectors. The two bacteriophages
Phage λ and M13 have been modified extensively and commonly used as cloning vectors

Bacteriophage lambda (λ) has been widely used in recombinant DNA since engineering of the
first viral cloning vector in 1974. Phage λ vectors are particularly useful for preparing genomic
libraries, because they can hold a larger piece of DNA than a plasmid vector. Today many
variations of λ vectors exist. Insertion vectors have unique restriction endonuclease sites that
allow the cloning of small DNA fragments in addition to the phage λ genome. These are often
used for preparing cDNA expression libraries. Replacement vectors have paired cloning sites on
either side of a central gene cluster. This central cluster contains genes for lysogeny and
recombination, which are not essential for the lytic life cycle (see Fig. 8.1).
The central gene cluster can be removed and foreign DNA inserted between the “arms.” All
phage vectors used as cloning vectors have been disarmed for safety and can only function in
special laboratory conditions.
A typical strategy for the use of a phage λ replacement vector is depicted in Fig. 8.8. The
recombinant viral particle infects bacterial host cells, in a process called “transduction.” The host
cells lyse after phage reproduction, releasing progeny virus particles. The viral particles appear
as a clear spot of lysed bacteria or “plaque” on an agar plate containing a lawn of bacteria. Each
plaque represents progeny of a single recombinant phage and contains millions of recombinant
phage particles. Most contemporary vectors carry a lacZ′ gene allowing blue-white selection.
Following are the advantages of phage cloning system over the Plasmids:
1. DNA can be packed in vitro into phage particles and transduced into E. coli with high
efficiency
2. Foreign DNA upto 25 kb in length can bee inserted into phage vector.
3. Screening and storage of recombinant DNA is easier.
RECOMBINANT DNA TECHNOLOGY TOOLS
The standard use of recombinant DNA technology was to add genetic information to the
genome, generating a gain of function in the resulting cells and transgenic organisms. Now that
such gene-transfer processes are routine, the focus of transgenic technology has shifted to the
provision of more control over the behavior of transgenes. New techniques have evolved in
parallel in animals and plants. Advances have come about through the development of inducible
expression systems that facilitate external regulation of transgenes and the exploitation of site-
specific recombination systems to make precise modifications in target genomes. In mice, the
combination of gene targeting, site-specific recombination and inducible transgene expression
makes it possible to selectively switch on and off both transgenes and endogenous genes in a
conditional manner. Other routes to gene silencing have also been explored, such as the
expression of antisense RNA and the recently described phenomenon of RNA interference.
Transgenic animals and plants are also being used increasingly as tools for the analysis of
genomes.
Moreover, the cloned genes are utilized commercially in medicals, agriculture and industry and
production of variable products.
An outline of these applications can be given as:

1. CLONED GENES AFOR PRODUCTION OF GROWTH HORMONES,

VACCINES AND COMMERCIAL CHEMICALS

HUMAN PEPTIDE HORMONE GENES

Insulin:
Itakura et al chemically synthesized DNA sequence for two chains A and B of insulin and were
separately inserted into two PBR322 plasmids. The recombinant plasmids were separately
transferred into E. coli cells which secreted fused β-galactosidase –B chain. These were isolated
and insulin was produced
Somatotropin (growth hormone)
Biosynthesis of Somatotropin was achieved through gene cloning procedures. For that double
stranded cDNAs were produced from mRNA precursor of hGH gene which was then
incorporated into bacterial cells where it was expressed in non-precursor form. The rDNA
plasmids are allowed to transform E. coli
Somatostatin
Two plasmids, PSOM 1 and PSOM 111 were constructed for the synthesis of Somatostatin. The
synthetic gene was introduced into E. coli and Somatostatin was produced.
B-endorphin
Shine et al integrated DNA sequences of B-endorphin obtained from mRNA adjacent to β-
galactosidase genes on plasmid.
HUMAN INTERFERON GENES
There are three types of human interferon , β and . These are being successfully produced
from genetically engineered E. coli cells
By isolation of mRNA from leukocytes and fibroblasts, production of cDNA, its integration into
PBR322 and incorporation and cloning into E. coli cells

GENES FOR VACCINES AND IMMUNOGENIC SUBSTANCES

Some of the vaccines synthesized by recombinant DNA technology are:
 Recombinant vaccine for Hepatitis B virus
 Indigenous Hepatitis B vaccine
 Vaccines for rabies virus
 Vaccines for polio virus
 Vaccines for foot and mouth disease virus
 Vaccines for small pox virus
 Malaria vaccines etc.

GENES ASSOCIATED WITH GENETIC DISEASES

Recombinant DNA technology is used to cure certain defective genetic diseases like
 Phenylketonuria genes
 Urokinase genes
 Thalassaemia genes
 Haemophilia genes

PRODUCTION OF COMMERCIAL CHEMICALS

There are several chemicals which are produced by using the recombinant DNA technologies. A
few are:
 Vitamins
 Organic Acids
 Alcohols
 Antibiotics

2. PRESERVATION, DIAGNOSIS AND CURE OF DISEASES

PRESERVATION OF DISEASES
For preservation of diseases several immunogenic polypeptides (vaccines) and proteins
(antibiotics) have been synthesized
DIAGNOSIS OF DISEASES
Such techniques have been solved the problem of conventional methods for diagnosis of many
diseases. DNA probe, monoclonal antibodies and antenatal diagnosis are some of methods used
for diagnosis of disease.
GENE THERAPY
A remarkable advancement in recombinant DNA technology has occurred and accumulated such
knowledge that has made possible to transfer genes for treatment of human diseases.
The abnormal gene expression can manifest in the form of specific genetic disorders. The
process of delivering of genes in humans to correct diseases is called as “Gene Therapy”
The ultimate goal of the gene therapy is the gene replacement therapy. Gene therapy permits
physiological regulation of the transgenes and elimination of other cellular genes which occur at
the time of random integration of foreign gene.
Gene therapy can be somatic therapy when genetic defects are corrected in somatic cells of the
body and germ-line gene therapy, when genetic defects are corrected in germ cell.

3. DNA FINGER PRINTING OR PROFILING

The complicated technology that facilitates the identification of individuals at genetic level is
known as DNA fingerprinting or DNA profiling. This genetic analysis is based on identification
of tiny segments of the hereditary material which testify the unique molecular signature which
cannot be altered. With the help of DNA profiling technology identify of burnt or unidentifiable
dead body can be done.

4. ANIMAL AND PLANT IMPROVEMENT

TRANSGENIC ANIAMLS
By the help of rDNA technology good yielding transgenic farm animals have been produced by
incorporation of different transgenes

CROP IMROVEMENT
Following are some steps towards crop improvement by rDNA technology:
 Transgenic plants
 Nif gene transfer
 Phaseolin gene transfer
 Conversion of C3 into C4 plants
 Herbicide resistant plants
 Insect pest resistant plants
 Plant improvement through genetic transformation
 Agro bacterium mediated gene transfer
RESTRICTION ENZYMES AND DNA LIGASES

Two major categories of enzymes are important tools in the isolation of DNA and the
preparation of recombinant DNA: restriction endonucleases and DNA ligases. Restriction
endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded DNA
molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere,
depending on the type of enzyme. DNA ligase joins two pieces of DNA by forming
phosphodiester bonds.

1. RESTRICTION ENZYMES
Restriction endonucleases are found in a wide range of bacterial species. Werner Arber
discovered in the early 1960s that their biological function is to recognize and cleave foreign
DNA (the DNA of an infecting virus, for example); such DNA is said to be restricted. In the host
cell’s DNA, the sequence that would be recognized by its own restriction endonuclease is
protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase.
The restriction endonuclease and the corresponding methylase are sometimes referred to as a
restriction-modification system.
Type I and III restriction endonucleases are not useful for gene cloning because they cleave
DNA at sites other than the recognition sites and thus cause random cleavage patterns. In
contrast, type II endonucleases are widely used for mapping and reconstructing DNA in vitro
because they recognize specific sites and cleave just at these sites (Table 8.1).
Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to
four nucleotides of one strand unpaired at each resulting end. These unpaired strands are referred
to as sticky ends (Fig. 9–3a), because they can base-pair with each other or with complementary
sticky ends of other DNA fragments.
Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester
bonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9–3b). The base-
pairing of complementary sticky ends greatly facilitates the ligation reaction (Fig. 9–3a). Blunt
ends can also be ligated, albeit less efficiently. Researchers can create new DNA sequences by
inserting synthetic DNA fragments (called linkers) between the ends that are being ligated.
Inserted DNA fragments with multiple recognition sequences for restriction endonucleases (often
useful later as points for inserting additional DNA by cleavage and ligation) are called
polylinkers (Fig. 9–3c).
Restriction endonclease nomenclature
Restriction endonucleases are named for the organism in which they were discovered, using a
system of letters and numbers. For example, HindIII (pronounced “hindee-three”) was
discovered in Haemophilusinfluenza (strain d). The Hin comes from the first letter of the genus
name and the first two letters of the species name; d is for the strain type; and III is for the third
enzyme of that type. SmaI is from Serratia marcescens and is pronounced “smah-one,” EcoRI
(pronounced “echo-r-one”) was discovered in Escherichia coli (strain R), and BamHI is from
Bacillus amyloliquefaciens (strain H). Over 3000 type II restriction endonucleases have been
isolated and characterized to date. Approximately 240 are available commercially for use by
molecular biologists.

2. DNA LIGASES
The study of DNA replication and repair processes led to the discovery of the DNA-joining
enzyme called DNA ligase. DNA ligases catalyze formation of a phosphodiester bond between
the 5′-phosphate of a nucleotide on one fragment of DNA and the 3′-hydroxyl of another (see
Fig. 6.14). This joining of linear DNA fragments together with covalent bonds is called ligation.
Unlike the type II restriction endonucleases, DNA ligase requires ATP as a cofactor.
Because it can join two pieces of DNA, DNA ligase became a key enzyme in genetic
engineering. If restriction-digested fragments of DNA are placed together under appropriate
conditions, the DNA fragments from two sources can anneal to form recombinant molecules by
hydrogen bonding between the complementary base pairs of the sticky ends. However, the two
strands are not covalently bonded by phosphodiester bonds. DNA ligase is required to seal the
gaps, covalently bonding the two strands and regenerating a circular molecule. The DNA ligase
most widely used in the lab is derived from the bacteriophage T4. T4 DNA ligase will also ligate
fragments with blunt ends, but the reaction is less efficient and higher concentrations of the
enzyme are usually required in vitro. To increase the efficiency of the reaction, researchers often
use the enzyme terminal deoxynucleotidyl transferase to modify the blunt ends. For example, if a
single-stranded poly (dA) tail is added to DNA fragments from one source, and a single stranded
poly (dT) tail is added to DNA from another source, the complementary tails can hydrogen bond
(Fig. 8.5). Recombinant DNA molecules can then be created by ligation.
 3. PHOSPHATASES
A Phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolyzing
phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group.
This action is directly opposite to that of phosphorylases and kinases, which attach phosphate
groups to their substrates by using energetic molecules like ATP.
A common phosphatase in many organisms is alkaline phosphatase. Another large group of
proteins present in archaea, bacteria and eukaryotes are deoxyribonucleotide and ribonuceotide
phosphatase or pyrophosphatase that catalyse the decomposition of dNTP/NTP in dNDP/NDP
and free phosphate ion ot dNMP/NMP and a free pyrophosphate ion. The other group of
phosphatase is collectively called as protein phosphatase, which removes a phosphate group
from the phosphorylated amino acid residue of the substrate protein. Protein phosphorylation is
common post translational modification of protein catalyzed by protein kinase, and protein
phosphatases reverse the effect
 4. T4 POLYNUCLEOTIDE KINASE
T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the  position of ATP to
the 5- hydroxyl terminus of double and single stranded DNA and RNA, as well as nucleoside 3-
mono phosphates.
Oligonucleotide labeling
Methods for end-labeling oligonucleotides do not involve DNA synthesis reactions. Instead, they
make use of other enzymes. In 5′ end-labeling, a g-phosphate from ATP is added to the 5′ end of
an oligonucleotide by the enzyme T4 polynucleotide kinase. In 3′ end-labeling, a labeled dNTP
is added to the 3′ end by the enzyme terminal transferase

5. KLENOW FRAGMENT OF DNA POLYMERASE I

It is the product of enzymatic breakdown of DNA pol I from E. coli. Its molecular weight is 76
Kd. It has 3′ → 5′ exonuclease activity. It has the ability to do polymerization under in vitro
conditions. It lacks 5′ → 3′ exonuclease activity

For most applications, including cloning of cDNAs, double stranded DNA is required. The
second DNA strand is generated by the Klenow fragment of DNA polymerase I from E. coli
(Fig. 1C). The 5′ → 3′ exonuclease activity of DNA polymerase I from E. coli makes it
unsuitable for many applications. However, this enzymatic activity can be readily removed from
the holoenzyme by exposure to a protease. The large or Klenow fragment of DNA polymerase I
generated by proteolysis has 5′ → 3′ polymerase and 3′ → 5′ exonuclease (proofreading)
activity, and is widely used in molecular biology. Commercially available Klenow fragments are
usually produced by expression in bacteria from a truncated form of the DNA polymerase I gene.
Klenow fill-in
A “fill-in” reaction is used to generate blunt ends on fragments created by cleavage with
restriction endonucleases that leave 5′ single-stranded overhangs. The Klenow fragment of E.
coli DNA polymerase I is used to fill in the gaps from 5′ to 3′, in the presence of dNTPs,
including one labeled dNTP (Fig.below). The result is a doublestranded DNA with the 3′ ends
labeled.
DNA SEQUENCING
Until the late 1970s, determining the sequence of a nucleic acid containing even five or ten
nucleotides was difficult and very laborious. The development of two new techniques in 1977,
one by Alan Maxam and Walter Gilbert (Chemical Degradation Method) and the other by
Frederick Sanger (enzymatic method/ dideoxy method) , has made possible the sequencing of ever
larger DNA molecules with an ease unimagined just a few decades ago. The techniques depend
on an improved understanding of nucleotide chemistry and DNA metabolism, and on
electrophoretic methods for separating DNA strands differing in size by only one nucleotide.
Electrophoresis of DNA is similar to that of proteins. Polyacrylamide is often used as the gel
matrix and in work with short DNA molecules (up to a few hundred nucleotides), Agarose is
generally used for longer pieces of DNA.
MAXAM AND GILBERT’S CHEMICAL DEGREDATIION METHOD
This method is not much popular because it is time consuming and labour intensive. In this
method, the DNA molecule can be radiolabelled at either 5 end by using polynucleotide kinase
or 3 end by terminal transferase. One end of radiolabelled double stranded DNA is removed
using endonuclease. A base is modified chemically follwed by cleavage of sugar-phosphate
backbone of DNA. Not any specific reaction for 4 bases is carried out, except specific reaction to
G only and purine specific reaction which removes A or G. a difference in these reactions
indicate the presence of A.
Cleavage of purine: the mixture is separated in four sets, each treated with different reagents
which degredate only G or C or A and G or C and T. in one set, DNA is treated with acid
followed by dimethyl sulphate. This causes methylation of A (at 3  position) and G ( at 7
position). Subsequently addition of alkali (-OH) and piperdine results in cleavage of DNA and
removal of purines (A or G)
Clevage of pyrimidines: similar to the cleavage of purine, pyrimidine (C or T) is also cleaved in
the presence of 1-2 M Nacl Soln. It works only with C. Difference b/w these two indicate the
presence of T in the DNA sequence cleavage of pyrimidines also takes place through hydrazine
hydrolysis.
Partial chemical cleavage of DNA fragments as done above generates the populations of
radiolabelled molecules extending from radiolabelled terminus to the site of chemical cleavage.
These fragments are of different sizes that represent unique pairs of 5 and 3 cleavage products
in the random collection. A complete set is formed by these products, the length of each no. is
short by one nucleotide. These can be separated by gel electrophoresis. The fragments containing
labelled terminus can be observed by autoradiography of the gel. Following the order of
fragments obtained from digestions the sequence of nucleotides is deduced and interpreted.
FREDERICK SANGER’S (ENZYMATIC METHOD/ DIDEOXY METHOD)
In this method, single-stranded DNA is mixed with a radioactively labeled primer to provide the
3′-OH required for DNA polymerase to initiate DNA synthesis. The primer is usually
complementary to a region of the vector just outside the multiple cloning sites. The sample is
then split into four aliquots, each containing DNA polymerase, four dNTPs (at high
concentration), and a low concentration of a replication terminator. The replication terminators
are dideoxynucleoside triphosphates (ddNTPs) that are missing the 3′-OH. Because they lack the
3′-OH, they cannot form a phosphodiester bond with another nucleotide. Thus, each reaction
proceeds until a replication-terminating nucleotide is added, and each of the four sequencing
reactions produces a series of single-stranded DNA molecules, each one base longer than the
last.
The polymerase of choice for DNA sequencing is phage T7 DNA polymerase (called
“Sequenase”). The sequencing mixtures are loaded into separate lanes of a denaturing
polyacrylamide gel and electrophoresis is used to separate the DNA fragments. Autoradiography
is used to detect a ladder of radioactive bands. The radioactive label (primer) is at the 5′ end of
each newly synthesized DNA molecule. Thus, the smallest fragment at the bottom of the gel
represents the 5′ end of the DNA. Reading the sequence of bases from the bottom up (5′ → 3′)
gives the sequence of the DNA molecule synthesized in the sequencing reaction. The sequence
of the original strand of DNA is complementary to the sequence read from the gel (3′ → 5′).
This method can separate DNA of approximately 500 nt. For longer sequences, overlapping
fragments are sequenced. The technique is very laborious and the sequences have to be read by
hand.
POLYMERASE CHAIN REACTION (PCR)

The polymerase chain reaction (PCR) is the one of the most powerful techniques that has been
developed recently in the area of recombinant DNA research. PCR has had a major impact on
many areas of molecular cloning and genetics. With this technique, a target sequence of DNA
can be amplified a billion-fold in several hours. Amplification of particular segments of DNA by
PCR is distinct from the amplification of DNA during cloning and propagation within a host cell.
The procedure is carried out entirely in vitro. In addition to its use in many molecular cloning
strategies, PCR is also used in the analysis of gene expression, forensic analysis where minute
samples of DNA are isolated from a crime scene and diagnostic tests for genetic diseases.

PCR is a DNA polymerase reaction. As with any DNA polymerase reaction it requires a DNA
template and a free 3′-OH to get the polymerase started. The template is provided by the DNA
sample to be amplified and the free 3′-OH groups are provided by site-specific oligonucleotide
primers. The primers are complementary to each of the ends of the sequence that is to be
amplified. Note that in vivo DNA polymerase would use an RNA primer, but a more stable, more
easily synthesized DNA primer is used in vitro. The three steps of the reaction are denaturation,
annealing of primers, and primer extension (Fig. 1):
 1. Denaturation. In the first step, the target sequence of DNA is heated to denature the
template strands and render the DNA single-stranded.
2. Annealing. The DNA is then cooled to allow the primers to anneal, that is, to bind the
appropriate complementary strand. The temperature for this step varies depending on the
size of the primer, the GC content, and its homology to the target DNA. Primers are
generally DNA oligonucleotides of approximately 20 bases each.
3. Primer extension. In the presence of Mg2+, DNA polymerase extends the primers on
both strands from 5′ to 3′ by its polymerase activity. Primer extension is performed at a
temperature optimal for the particular polymerase that is used. Currently, the most
popular enzyme for this step is Taq polymerase, the DNA polymerase from the
thermophilic (heat-loving) bacteria Thermus aquaticus. This organism lives in hot springs
that can be near boiling and thus requires a thermostable polymerase.
These three steps are repeated from 28 to 35 times. With each cycle, more and more fragments
are generated with just the region between the primers amplified. These accumulate
exponentially. The contribution of strands with extension beyond the target sequence becomes
negligible since these accumulate in a linear manner. After 25 cycles in an automated
thermocycler machine, there is a 225 amplification of the target sequence. PCR products can be
visualized on a gel stained with nucleic acid-specific fluorescent compounds such as ethidium
bromide or SYBR green. The error rate of Taq is 2 × 10−4. If an error occurs early on in the
cycles, it could become prominent. Other polymerases, such as Pfu, have greater fidelity. Pfu
DNA polymerase is from Pyrococcus furiosus. Base misinsertions that may occur infrequently
during polymerization are rapidly excised by the 3′ → 5′ exonuclease (proofreading) activity of
this enzyme.
When Kary Mullis first developed the PCR method in 1985, his experiments used E. coli DNA
polymerase. Because E. coli DNA polymerase is heat-sensitive, its activity was destroyed during
the denaturation step at 95°C. Therefore, a new aliquot of the enzyme had to be added in each
cycle. The purification, and ultimately the cloning, of the DNA polymerase from T. aquaticus
made the reaction much simpler. In his first experiments, Mullis had to move the reaction
manually between the different temperatures. Fortunately, this procedure has been automated by
the development of thermal cyclers. These instruments have the capability of rapidly switching
between the different temperatures that are required for the PCR reaction. Thus the reactions can
be set up and placed in the thermal cycler, and the researcher can return several hours later (or
the next morning) to obtain the products.
APPLICATIONs OF PCR
PCR is used in:
 Diagnosis of pathogens
 Diagnosis of specific mutations (sickle cell anaemia, phenylketonuria and muscular
dystrophy)
 Prenatal diagnosis of several genetic diseases
 DNA fingerprinting
 Diagnosis of plant diseases
 Sexing of embryo (sex of human or livestock embryos, Fertilized embryos)
 Molecular archaeology (palaentology)
 molecular cloning strategies,
 Analysis of gene expression,
 forensic analysis
CONSTRUCTING DNA LIBRARIES
Vectors are used to compile a library of DNA fragments that have been isolated from the
genomes of a variety of organisms. This collection of fragments can then be used to isolate
specific genes and other DNA sequences of interest. DNA fragments are generated by cutting the
DNA with a specific restriction endonuclease. These fragments are ligated into vector molecules,
and the collection of recombinant molecules is transferred into host cells, one molecule in each
cell. The total number of all DNA molecules makes up the library. This library is searched, that
is screened, with a molecular probe that specifically identifies the target DNA. Once prepared the
library can be perpetuated indefinitely in the host cells and is readily retrieved whenever a new
probe is available to seek out a particular fragment. Two main types of libraries can be used to
isolate specific DNAs: genomic and cDNA libraries.

Genomic library
A genomic library contains DNA fragments that represent the entire genome of an organism. The
first step in creating a genomic library is to break the DNA into manageable size pieces (e.g. 15–
20 kb for phage λ vectors), usually by partial restriction endonuclease digest. Under limiting
conditions, any particular restriction site is cleaved only occasionally, so not all sites are cleaved
in any particular DNA molecule. This generates a continuum of overlapping fragments. The
second step is to purify fragments of optimal size by gel electrophoresis or centrifugation
techniques. The final step is to insert the DNA fragments into a suitable vector. In humans, the
genome size is approximately 3 × 109 bp. with an average insert size of 20 kb, the number of
random fragments to ensure with high probability (95–99%) that every sequence is represented is
approximately 106 clones for humans. The maths actually works out to 1.5 × 105 (i.e. (3 × 109
bp)/(2 × 104 bp) but more clones are needed in practice, since insertion is random. Bacteriophage
 or cosmid vectors are typically used for genomic libraries. Since a larger insert size can be
accommodated by these vectors compared with plasmids, there is a greater chance of cloning a
gene sequence with both the coding sequence and the regulatory elements in a single clone.

cDNA library
The principle behind cDNA cloning is that an mRNA population isolated from a specific tissue,
cell type, or developmental stage (e.g. embryo mRNA) should contain mRNAs specific for any
protein expressed in that cell type or during that stage, along with “housekeeping” mRNAs that
encode essential proteins such as the ribosomal proteins, and other mRNAs common to many
cell types or stages of development. Thus, if mRNA can be isolated, a small subset of all the
genes in a genome can be studied. mRNA cannot be cloned directly, but a cDNA copy of the
mRNA can be cloned. Because a cDNA library is derived from mRNA, the library contains the
coding region of expressed genes only, with no introns or regulatory regions. This latter point
becomes important for applications of recombinant DNA technology to the production of
transgenic animals and for human gene therapy
COMPLEMENTARY DNA (cDNA) SYNTHESIS
A cDNA fragment is prepared directely by using mRNA as tempelate. It involves following
steps:
Isolation of mRNA
Most eukaryotic mRNAs are poly-adenylated at the 3′ end to form a poly(A) tail. This has an
important practical consequence that has been exploited by molecular biologists. The poly(A)
region can be used to selectively isolate mRNA from total RNA by affinity chromatography
(Fig. 1A). The purified mRNA can then be used as a template for synthesis of a complementary
DNA (cDNA).
Purification of mRNA
Total RNA is extracted from a specific cell type that expresses a specific set of genes. Of this
total cellular RNA, 80–90% is rRNA, tRNA, and histone mRNA, not all of which have a poly
(A) tail. These RNAs can be separated from the poly (A) mRNA by passing the total RNA
through an affinity column of oligo(dT) or oligo(U) bound to resin beads. Under conditions of
relatively high salt the poly (A) RNA is retained by formation of hydrogen bonds with the
complementary bases, and the RNA lacking a poly(A) tail flows through.
The salt conditions for hybridization are similar to the ion concentration in cells (e.g. 0.3–0.6 M
NaCl). The poly(A) mRNA is then eluted from the column in low salt elution buffer (e.g. 0.01 M
NaCl), which promotes denaturation of the hybrid.
First strand synthesis of cDNA (Reverse transcription)
A number of strategies can be used to synthesize cDNA from purified mRNA. One strategy is as
follows. In brief, cDNA is synthesized by the action of reverse transcriptase and DNA
polymerase (Fig. 1B). The reverse transcriptase catalyzes the synthesis of a single-stranded DNA
from the mRNA template. Like a regular DNA polymerase, reverse transcriptase also needs a
primer to get started. A poly(dT) primer is added to provide a free 3′-OH end that can be used for
extension by reverse transcriptase in the presence of deoxynucleoside triphosphates (dNTPs).
Usually a viral reverse transcriptase is employed such as one from avian myeloblastosis virus
(AMV). The reverse transcriptase adds dNTPs from 5′ to 3′ by complementary base pairing. This
is called first strand synthesis. The mRNA is then degraded with a ribonuclease or an alkaline
solution.
Second strand synthesis of cDNA
For most applications, including cloning of cDNAs, doublestranded DNA is required. The
second DNA strand is generated by the Klenow fragment of DNA polymerase I from E. coli
(Fig. 1C). The 5′ → 3′ exonuclease activity of DNA polymerase I from E. coli (see Focus box
6.1) makes it unsuitable for many applications. However, this enzymatic activity can be readily
removed from the holoenzyme by exposure to a protease. The large or Klenow fragment of DNA
polymerase I generated by proteolysis has 5′ → 3′ polymerase and 3′ → 5′ exonuclease
(proofreading) activity, and is widely used in molecular biology. Commercially available
Klenow fragments are usually produced by expression in bacteria from a truncated form of the
DNA polymerase I gene.
There is a tendency for the reverse transcriptase enzyme used in first strand synthesis to loop
back on itself and start to make another complementary strand. This hairpin forms a natural
primer for DNA polymerase and a second strand of DNA is generated. S1 nuclease (from
Aspergillus oryzae) is then added to cleave the single-stranded DNA hairpin. Double-strand
DNA linkers with ends that are complementary to an appropriate cloning vector are added to the
double-strand DNA molecule before ligation into the cloning vector. The end result is a double-
stranded cDNA in which the second strand corresponds to the sequence of the mRNA, thus
representing the coding strand of the gene. The sequences that appear in the literature are the 5′
→ 3′ sequences of the second strand cDNA (Fig. 1). Sequences corresponding to introns and to
promoters and all regions upstream of the transcriptional start site are not represented in cDNAs.
The library created from all thecDNAs derived from the mRNAs in the specific cell type forms
the cDNA library of cDNA clones.
Real-time PCR
Real time polymerase chain reaction is a technique laboratory technique of Molecular Biology
based on PCR. It monitors the amplification of targeted DNA molecule during PCR i.e in real
time not in its end, as in conventional PCR.
There are many applications of the PCR where it would be advantageous to be able to quantify
the amount of starting material. Theoretically, there is a quantitative relationship between the
amount of starting material (target sequence) and the amount of PCR product at any given cycle.
In practice, replicate reactions yield different amounts of product, making quantitation
unreliable. Higuchi et al. (1992, 1993) pioneered the use of ethidium bromide to quantify PCR
products as they accumulate. Amplification produces increasing amounts of double stranded
DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the
increase in fluorescence versus cycle number it is possible to analyse the PCR kinetics in real
time. This is much more satisfactory than analysing product accumulation after a fixed number
of cycles.
The principal drawback to the use of ethidium bromide is that both specific and non-specific
products generate a signal. This can be overcome by the use of probe-based methods for assaying
product accumulation (Livak et al. 1995). The probes used are oligonucleotides with a reporter
fluorescent dye attached at the 5′ end and a quencher dye at the 3′ end. While the probe is intact,
the proximity of the quencher reduces the fluorescence emitted by the reporter dye. If the target
sequence is present, the probe anneals downstream from one of the primer sites. As the primer is
extended, the probe is cleaved by the 5′ nuclease activity of the Taq polymerase (Fig). This
cleavage of the probe separates the reporter dye from the quencher dye, thereby increasing the
reporter-dye signal. Cleavage removes the probe from the target strand, allowing primer
extension to continue to the end of the template strand. Additional reporter-dye molecules are
cleaved from their respective probes with each cycle, affecting an increase in fluorescence
intensity proportional to the amount of amplicon produced.
Instrumentation has been developed which combines thermal cycling with measurement of
fluorescence, thereby enabling the progress of the PCR to be monitored in real time. This
revolutionizes the way one approaches PCR-based quantitation of DNA. Reactions are
characterized by the point in time during cycling when amplification of a product is first
detected, rather than by the amount of PCR product accumulated after a fixed number of cycles.
The higher the starting copy number of the target, the sooner a significant increase in
fluorescence is noted. Quantitation of the amount of target in unknown samples is achieved by
preparing a standard curve, using different starting copy numbers of the target sequence.
 FIG. REAL TIME PCR

MOLECULAR MARKERS
Molecular Marker also called as genetic Marker is a gene or DNA sequence with a known
location on chromosome that can be used to identify individuals or species. It can be described as
a variation (which may arise due to mutation or alteration in genome loci) that can be observed, a
Genetic Marker may be short DNA sequence such as a sequence surrounding a single bp change
(single nucleotide polymorphism-SNP) or a long one like mini-satellites.
Some commonly used types of Genetic Markers are:
 RFLP (restriction fragment length polymorphism)
 AFLP (Amplified fragment length polymorphism)
 SSLP (Simple Sequence length polymorphism)
 RAPD (Random Amplification Polymorphic DNA)
 VNTR (Variable Number Tandom Repeats)
  SSR (Simple Sequence Repeats)
 SNP (Single Nucleotide Polymorphism)
 STR (Short Tandom Repeats)
 SFP (Single Feature Nucleotide)
 DAT (Diversity Arrays Technology)
 RAD (Restriction site associated DNA markers)

Genetic Markers are used to know the relationship b/w an inherited disease and its genetic cause.

RFLP – AS GENETIC MARKER

In 1980, Mark Skolnick, Ray White, David Botstein, and Ronald Davis created a restriction
fragment length polymorphism (RFLP, pronounced “rif-lip”) marker map of the human genome.
A RFLP is defined by the existence of alternative alleles associated with restriction fragments
that differ in size from each other. RFLPs are visualized by digesting DNA from different
individuals with restriction endonucleases, followed by gel electrophoresis to separate fragments
according to size, then Southern blotting and hybridization to a labeled probe that identifies the
locus under investigation. A RFLP is demonstrated whenever the Southern blot pattern obtained
with one individual is different from the one obtained with another individual. These variable
regions do not necessarily occur in genes, and the function of most of those in the human
genome is unknown. An exception is a RFLP that can be used to diagnose sickle cell anemia
(Fig. 8.14). In individuals with sickle cell anemia, a point mutation in the β-globin gene has
destroyed the recognition site for the restriction endonuclease MstII. This mutation can be
distinguished by the presence of a larger restriction fragment on a Southern blot in an affected
individual, compared with a shorter fragment in a normal individual.
AFLP – AS GENETIC MARKER
AFLP is a PCR based tool used in genetic research, DNA fingerprinting and in practice of
genetic engineering. It has been developed in 1990s by Keygene. It involve:
 Restriction endonuclease digestion of genomic DNA
 Ligation of specific adapters
 Amplification of the restriction fragments by PCR using primer pairs containing common
sequences of the adapter and two or three arbitrary nucleotides and finally
 Analysis of the amplified fragments using gel electrophoresis

The amplified fragments are separated and visualized on denaturing polyacrylamide gels
through autoradiography or via fluorescence methodologies or via automated capillary
sequencing instruments.
The AFLP technology has the capacity to detect various polymorphisms in different genomic
regions simultaneously. It is also highly sensitive and reproducible, as a result, AFLP has
become widely used for the identification of genetic variation in strains or closely related species
of plants, feungi, animals and bacteria
RNA SILENCING, ANTISENSE RNA AND RNA INTERFERENCE
RNA silencing and RNA interference
In higher eukaryotes, including nematodes, fruit flies, plants, and mammals, a class of small
RNAs has been discovered that mediates the silencing of particular genes. The RNAs function
by interacting with mRNAs, often in the 3 UTR, results in either mRNA degradation or
translation inhibition. In case, the mRNA, and thus the gene that produces it, is silenced. This
form of gene regulation controls developmental timing in at least some organisms. It is also used
as a mechanism to protect against invading RNA viruses (particularly important in plants, which
lack an immune system) and to control the activity of transposons. In addition, small RNA
molecules may play a critical (but still undefined) role in the formation of heterochromatin.
The small RNAs are sometimes called micro-RNAs (miRNAs). Many are present only
transiently during development, and these are sometimes referred to as small temporal RNAs
(stRNAs). Hundreds of different miRNAs have been identified in higher eukaryotes. They are
transcribed as precursor RNAs about 70 nucleotides long, with internally complementary
sequences that form hairpinlike structures (Fig below). The precursors are cleaved by
endonucleases to form short duplexes about 20 to 25 nucleotides long. The best-characterized
nuclease goes by the delightfully suggestive name Dicer; endonucleases in the Dicer family are
widely distributed in higher eukaryotes. One strand of the processed miRNA is transferred to the
target mRNA (or to a viral or transposon RNA), leading to inhibition of translation or
degradation of the RNA (Fig.below).
This gene regulation mechanism has an interesting and very useful practical side. If an
investigator introduces into an organism a duplex RNA molecule corresponding in sequence to
virtually any mRNA, the Dicer endonuclease cleaves the duplex into short segments, called
small interfering RNAs (siRNAs). These bind to the mRNA and silence it. The process is known
as RNA interference (RNAi). In plants, virtually any gene can be effectively shut down in this
way. In nematodes, simply introducing the duplex RNA into the worm’s diet produces very
effective suppression of the target gene. The technique has rapidly become an important tool in
the ongoing efforts to study gene function, because it can disrupt gene function without creating
a mutant organism. The procedure can be applied to humans as well. Laboratory-produced
siRNAs have already been used to block HIV and poliovirus infections in cultured human cells
for a week or so at a time. Although this work is in its infancy, the rapid progress makes RNA
interference a field to watch for future medical advances.
RNA interference (RNAi) is a novel phenomenon that has the potential to become an extremely
powerful tool for gene silencing in any organism. The process was discovered by Fire et al.
(1998) while investigating the use of antisense and sense RNA for gene inhibition in the
nematode worm Caenorhabditis elegans. In one experiment, they introduced both sense and
antisense RNA into worms simultaneously and observed a striking and specific inhibitory effect,
which was approximately 10-fold more efficient than either single RNA strand alone.
The phenomenon of RNAi appears to be quite general, and this strategy has been used more
recently for gene silencing in many other organisms, including Drosophila, mice and plants. In
C. elegans, RNAi is now the standard procedure for gene inactivation, and it is becoming
increasingly favoured in Drosophila and plants, due to its potency and specificity.
Like post-transcriptional gene silencing (PTGS) in plants, RNAi is also systemic. RNAi-
mediated silencing can be achieved in C. elegans by injecting dsRNA into any part of the body,
but, more remarkably, simply placing worms in a solution containing dsRNA or feeding them on
bacteria that synthesize dsRNA is sufficient to trigger the effect. The similarities between RNAi
and PTGS in plants suggest a common molecular basis.

Antisense RNA
Antisense RNA has the opposite sense to mRNA. The presence of complementary sense and
antisense RNA molecules in the same cell can lead to the formation of a stable duplex, which
may interfere with gene expression at the level of transcription, RNA processing or possibly
translation. Antisense RNA is used as a natural mechanism to regulate gene expression in a
number of prokaryote systems and, to a lesser extent, in eukaryotes. Transient inhibition of
particular genes can be achieved by directly introducing antisense RNA or antisense
oligonucleotides into cells. However, the transformation of cells with antisense transgenes (in
which the transgene is inverted with respect to the promoter) allows the stable production of
antisense RNA and thus the long-term inhibition of gene expression. This principle was
established in transgenic animals and plants at about the same time. Katsuki et al. (1988)
constructed an expression cassette in which the mouse myelin basic protein (MBP) cDNA was
inverted with respect to the promoter, thus producing antisense RNA directed against the
endogenous gene. In some of the transgenic mice, there was up to an 80% reduction in the levels
of MBP, resulting in the absence of myelin from many axons and generating a phenocopy of the
myelin-depleted ‘shiverer’ mutation..
Antisense constructs have been widely used in transgenic animals and plants for gene inhibition.
However, the efficiency of the technique varies widely and the effects can, in some cases, be
non-specific. In some experiments, it has been possible to shutdown endogenous gene activity
almost completely,

ETHICS AND BIOSAFETY OF DNA RECOMBINANT TECHNOLOGY

Paul Berg shared the 1980 Nobel Prize in chemistry for creating the first cloned DNA molecule,
a hybrid  phage that contained the genome of the simian tumor virus, SV40. The fact that he
could do this work was worrisome to many people, included him. The recombinant DNA dispute
was underway. Berg voluntarily stopped inserting tumor virus genes into phages that attack the
common intestinal bacteria E. coli.
People continue to worry about the dangers of working with recombinant DNA. One immediate
and obvious concern is that cancer or toxin genes will “escape” from the laboratory. In other
words, recombinant DNA technology could create a bacterium or plasmid that contained toxin or
tumor genes. The modified bacterium or plasmid could then accidentally infect people. A 1974
report by the National Academy of Sciences led to a February 1975 meeting, which took place at
the Asilomar Conference Center south of San Francisco. Berg convened this meeting, which
over one hundred molecular biologists attended. The recommendations of the Asilomar
Committee later formed the basis for official guidelines developed by the National Institutes of
Health (NIH). In essence, NIH established guidelines of containment.
Containment means erecting physical and biological barriers to the escape of dangerous
organisms. The NIH guidelines defined four levels of risk, from minimal to high, and four levels
of physical containment for them (called P1 through P4). The most hazardous experiments,
dealing with the manipulation of tumor viruses and toxin genes, require extreme care, which
included negative pressure air locks to the laboratory and experiments done in laminar-flow
hoods, with filtered or incinerated exhaust air.
Current concern is focused on the acceptability of genetically modified crops (GM crops). As we
will discuss later, one fourth of American cropland is planted with genetically modified crops,
modified mainly for insect resistance. These modifications have curtailed our use of insecticides.
(For cotton and corn, for example, liquid insecticide use dropped by 3.6 million liters and
powdered insecticide by 300,000 kilograms in 1999.) However, people are concerned with the
effects these modifications might have on natural ecosystems: How many valuable insects will
be killed by mistake? Although Third World countries are desperate for these technologies, the
United States, European and Asian trading partners are demanding that the crops we export be
genetically unmodified. Farmers are also concerned that genetically modified crops have been
modified to be sterile (so called “terminator technology”) so that farmers would need to buy new
seeds each year.
More recently, the recombinant DNA dispute has taken a whole new twist. It now has surfaced
as a conflict between academic freedom and industrial secrecy. It seems that recombinant
DNA technology is very lucrative. Numerous academic scientists have either begun genetic
engineering companies or become affiliated with pharmaceutical companies. However, the
philosophies of private enterprise and academia are often in conflict. Academic endeavors are
presumably open, with free exchange of information among colleagues, whereas private
enterprise entails some degree of secrecy, at least until patents are obtained to protect the
investments of the companies. Thus, a basic conflict can arise for scientists trained in gene
cloning. The conflict has been prevalent since late 1980, when the first patent for recombinant
DNA techniques was awarded to Stanford University and the University of California. When, in
April 2000, United States President Bill Clinton and British Prime Minister Tony Blair issued a
joint statement asking that human genome data not be patented, the American stock market took
a major downturn. This is a tumultuous time for biotechnology.

Genetically modified food controversies

There is controversy over GMOs, especially with regard to their use in producing food. The
dispute involves buyers, biotechnology companies, governmental regulators, non-governmental
organizations, and scientists. The key areas of controversy related to GMO food are whether GM
food should be labeled, the role of government regulators, the effect of GM crops on health and
the environment, the effect on pesticide resistance, the impact of GM crops for farmers, and the
role of GM crops in feeding the world population. In 2014, sales of products which had been
labeled as non-GMO grew 30 percent to $1.1 billion.
There is a general scientific agreement that food from genetically modified crops is not
inherently riskier to human health than conventional food. No reports of ill effects have been
proven in the human population from ingesting GM food.

Although labeling of GMO products in the marketplace is required in many countries, it is not
required in the United States and no distinction between marketed GMO and non-GMO foods is
recognized by the US FDA. In a May 2014 article in The Economist it was argued that, while
GM foods could potentially help feed 842 million malnourished people globally, laws such as
those being considered by Vermont's governor, Peter Shumlin, to require labeling of foods
containing genetically modified ingredients, could have the unintended consequence of
interrupting the benign process of spreading GM technologies to impoverished countries that
suffer with food security problems.