Last Update: 2 November 2017

Part I
M - 31
Recombinant DNA technology and Genetic Engineering

Concept: (Plasmid, engineering the plasmid, getting plasmid into the bacteria, screening the
bacterial colonies, hybridization, making a probe, cloning and gene libraries.)
Ref: Microbiology- Prescott, Genetics- Russell, Klug& Cummings, Biotechnology-Meyers.

Genetic Engineering or gene technology is a particular field of biotechnology which

involves alteration of genetic constitution of cells or individuals (genetic manipulation)
by the selective removal, insertion, or modification of individual gene sets. The product
is thus obtained is recombinant DNA. (The term recombinant DNA refers to the creation
of a new combination of DNA molecules that are not found together naturally.)

Although human beings have altering the genetic makeup of organisms for centuries by
selective breeding, only recently has the direct manipulation of DNA been possible. The
deliberate modification of an organisms genetic information by directly changing its nucleic
acid genome is called genetic engineering and is accompanied by a collection of methods
known as recombinant DNA technology.
The basic procedures involve a series of steps:
1. First, the DNA responsible for a particular phenotype is identified and purified from
cells or tissues (isolation of gene).
2. Once purified, the gene(s) are fused with other pieces of DNA (mostly the plasmid) to
form recombinant DNA(r DNA) molecules.
3. These are propagated (gene cloning) by insertion into an organism (the host cell) that
need not even be in the same kingdom as the original gene donor. Within the host cell,
the recombinant molecule replicates, producing dozens of identical copies known as
clones. (Clones are identical organisms, cells, or molecules descended from a single
ancestor.)
4. Production of expression vector (mostly an E. coli strain) for cloned gene to express
the foreign gene where the cloned gene can be transcribed, its mRNA translated, and the
gene product isolated and used for research, or sold commercially.

In addition, DNA sequences up to about 100 bases long can now be chemically synthesized
by entirely automated procedures. Recombinant DNA molecules thus can be produced
containing either natural DNA fragments resulting from restriction-enzyme cleavage or any
desired chemically synthesized mutant sequences.

Thus it is an essential part of biotechnology, which is now experiencing a stage of

exceptionally rapid growth and development. Although the term has several definitions, in

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this text biotechnology refers to those processes in which living organisms are manipulated,
particularly at the molecular genetic level, to form useful products.

Historical Perspectives: Recombinant DNA technology is very much the result of several
key discoveries in microbial genetics. These are as follows--
In the late 1960s one of the first breakthroughs leading to recombinant DNA technology
was the discovery of microbial enzymes (restriction enzymes or restriction
endonucleases) by Werner Arber and Hamilton Smith (in 1978 Nobel Prize was awarded
to them for this discovery) that make cuts in double stranded DNA about 4 to 6 base pairs
long. Restriction enzymes can be used to prepare DNA fragments containing specific
genes or portions of genes.
In 1970 Howard Temin and David Baltimore independently discovered the enzyme
reverse transcriptase that retroviruses use to produce DNA copies of their RNA genome.
This enzyme can be used to construct a DNA copy, called complementary DNA
(cDNA), of any RNA. Thus genes or major portions of genes can be synthesized from
mRNA.
The next advance came in 1972, when David Jackson, Robert Symons, and Paul Berg
reported that they had successfully generated recombinant DNA molecules.
Within a year, plasmid vectors, or carriers of foreign DNA fragments during gene
cloning, had been developed and combined with foreign DNA. The first such
recombinant plasmid capable of being replicated within a bacterial host was the pSC101
plasmid constructed by Stanley Cohen and Herbert Boyer in 1973(SC in the plasmid
name stands for Stanley Cohen).
In 1975 Edwin M. Southern published a procedure, called Southern blotting technique
for detecting specific DNA fragments so that a particular gene could be isolated from a
complex DNA mixture.
By the late 1970s techniques for easily sequencing DNA, synthesizing oligonucleotides,
expressing eukaryotic genes in prokaryotes (expression vectors) had also been developed.

These techniques could then be used to solve practical problems. In 1982 commercial
production of genetically engineered human insulin by E.coli started.

Important points is recombinant DNA technology:

RESTRICTION ENZYMES
The cornerstone of recombinant DNA technology is a class of enzymes called restriction
endonucleases. These enzymes, isolated from bacteria , received their name because they
restrict or prevent viral infection by degrading the invading viral DNA. Restriction enzymes
recognize a specific sequence and cut both strands of the DNA within the sequence. To date,
over 200 restriction enzymes have been identified. Their usefulness in cloning derives from
their ability to reproducibly cut DNA into fragments.
One of the first restriction enzymes identified was isolated from E. coli by Herbert Boyer in
1969 and was designated EcoRI ( pronounced echo-r-one), cleaves the DNA between G and
A in the base sequence GAATTC. Note that in the double stranded condition , the base
sequence GAATTC will base pair with the same sequence running in the opposite direction

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( The sequence that read the same in both directions are known as palindromes.). EcoRI
therefore cleaves both strands between the G and A. When the two DNA fragments separate,
they contains single stranded complementary ends, known as sticky ends or cohesive ends
that can reanneal with complementary single stranded tails on other DNA fragments. If they
are mixed together under the proper conditions, DNA fragments from these two sources can
form recombinant molecules by hydrogen bonding of their sticky ends. The enzyme DNA
ligase can be used to covalently link these fragments to form recombinant DNA molecules.

----------- G-A-A-T-T-C--------- -----------G-A-A-T-T-C--------

----------- C-T-T-A-A-G-------- ----------- C-T-T-A-A-G-------

Cleavage with EcoRI Cleavage with EcoRI

----------G A-A-T-T-C-------
----------C-T-T-A-A G------
Fragments with complementary
tails

Gap Anneling allows recombinant

DNA molecules to form by
--------------G A-A-T-T-C------------- complementary base pairing .
--------------C-T-T-A-A G-------------- The two strands are not covalently
bonded as indicated by gaps.
Gap

DNA ligase

-------------G-A-A-T-T-C------------- DNA ligase seals the gaps

-------------C-T-T-A-A-G------------- covalently bonding the two
strands
Fig: Formation of recombinant DNA

Other restriction enzymes such as SmaI cleave DNA to produce blunt end fragments. DNA
fragments with blunt ends can also be joined together to create recombinant DNA molecules,
after the DNA has been modified. The enzyme terminal deoxynucleotide transferase is used
to create single-stranded ends by the addition of nucleotide tails. If a poly-dA tail is added
to DNA fragments from one source, and a poly-dT tail is added to DNA from another source,
complementary tails are created, and the fragments can reanneal by hydrogen bond
formation. Recombinant molecules can then be created by ligation.

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 Eukaryotic DNA Plasmid DNA

5-----------C-C-C G-G-G----------3
3-----------C-C-C C-C-C----------5

add poly-dT tails add poly-dA tails

Terminal deoxynucleotidyl transferase

5-----------C-C-C A-A-A-G-G-G--------3
3-----------G-G-G-T-T-T C-C-C--------5

Gap

5-------------C-C-C A-A-A-G-G-G-------------3 Annealing of

3------------G-G-G-T-T-T C-C-C---------------5 fragments

Gap

Ligation with DNA ligase

5-------------C-C-C- A-A-A-G-G-G-------------3
3------------G-G-G-T-T-T- C-C-C---------------5

Fig: Formation of recombinant DNA molecule by blunt cut

Table : Some Restriction Endonucleases and Their Recognition Sequences

Enzyme Microbial Sources Sequences

AluI Arthrobacter lut 5--A--G--C--T--3

3--T--C--G--A5

BamHI Bacillus amyloliquefaciens H 5GGATCC3

3CCTAGG--5

EcoRII E. coli 5CCTGG3

3GAACC5

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HaeIII Haemophilus aegyptius 5GGCC3
3CCGG5

HindIII Haemophilus influenzae b 5AA--GCTT3

3TTCGAA5

TaqI Thermus aquaticus 5TCGA3

3AGCT5

All restriction endonucleases conduct the double- strand, Mg+2 catalyzed hydrolysis of the
phosphodiester internucleotide linkage of DNA. In contrast, DNA modification
methyltransferase, or methylases, catalyze a fundamentally different reaction using the
same DNA sequence as substrate. The reaction produces the methylated DNA , abbreviated
AdoHcy.
These enzymes are organized into three distinct classes, based on the structures of the
proteins themselves, the cleavage and methylation mechanisms, and the nucleotide
sequences recognized. The categories are called, simply, typesI, II, and III; a fourth,
emergent, class, the methyl-dependent systems McrA, McrBC, and Mrr, consists of enzymes
whose sequence specificities remain incompletely determined. Type I and III enzymes are
similar in that their restriction and methylation components are different subunits of the same
holoenzyme, whereas the type II enzymes consist of independent enzymes. Type II
restriction-modification enzymes are simplest in terms of structure and function. The
endonuclease cleaves normally within or adjacent to a palindromic recognition sequence of
four to eight base pairs, with the sole requirement of Mg+2 as cofactor. Some type II
enzymes, designated type IIS , recognize non-palindromic sequences and cleave at defined
positions outside the sequence.
HYBRIDIZATION AND SOUTHERN BLOTTING
Hybridization is the formation of partially or completely double stranded (duplex) nucleic
acid (DNA: DNA, DNA: RNA, or RNA: RNA) by sequence specific interaction of two
complementary single stranded nucleic acids. Reassociation andrenaturation are terms
often used to describe hybridization between completely complementary DNA or RNA
strands. Solution hybridization is an integral part of the polymerase chain reaction.
Hybridization is often carried out using a labeled probe in an attempt to detect the existence
and quantity of complementary sequence in a complex nucleic acid mixture. The target is
often immobilized, as in Southern and other blotting techniques.

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The Southern blotting technique depends on the specificity of base complementarity in
nucleic acids. DNA fragments produced by restriction endonuclease digestion are first
separated by size with agarose gel electrophoresis. The DNA in the gel is denatured into
single- stranded fragments by treatment with an alkaline solution, and transferred to a sheet
of DNA-binding material, usually nitrocellulose or a nylon membrane so that each fragment
is firmly bound to the filter as the same position as the gel. To transfer the fragments, the
sheet of membrane is placed on the top of the gel, and a buffer solution flows through the gel
and the membrane by capillary action. As the buffer solution flows through both, the DNA
fragments move out of the gel and become immobilized on the membrane.
The DNA fragments bound to the membrane are hybridized with a labeled single-stranded
DNA probe (A short, labeled nucleic acid segment complementary in base sequence to
part of another nucleic acid, which is used to identify or isolate the particular nucleic acid

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from a mixture through its ability to bind specifically with the target nucleic acid). Only
the DNA fragments bound to the membrane that are complementary to the nucleotide
sequence of the probe will form double stranded hybrids. The unbound probe is washed
away, and the hybridized fragments are visualized by autoradiography. In this technique a
sheet of photographic film is placed over the filter for several hours and then developed. The
film is exposed and becomes dark everywhere a radioactive fragment is located because the
energy released by the isotope cause the formation of dark- silver grains.
Using Southern blotting, one can detect and isolate fragments with any desired sequence
from a complex mixture. More recently, non-radioactive probes to detect specific DNAs
have been developed. In one approach the DNA probe is linked to an enzyme such as
horseradish peroxidase (HRP). After the enzyme-DNA probe has bound to a DNA fragment
on the filter, the substrate luminol that will emit light when acted on by the peroxidase is
added. The chemiluminescent probe is detected by exposing the filter to photographic film
for about 20 mins. A second technique makes use of the vitamin biotin. A biotin DNA
probe is detected by incubating the filter with either the protein avidin or a similar bacterial
protein, streptavidin. The protein specifically attaches to biotin, and is visualized with a
special reagent containing biotin complexed with the enzyme alkaline phosphatase. The
bands with the probe appear blue. These nonradioactive techniques are more rapid and safer
than using radioisotopes. On the other hand, they may be less sensitive than radioactively
labeled probes.
In addition to characterizing cloned DNAs, Southern blots are used for many other
purposes, including the mapping of restriction sites within and near a gene, the identification
of DNA fragments carrying a single gene from a mixture of many fragments, and the
identification of related genes in different species. Southern blots are also used to detect
rearrangements and duplications in genes associated with human genetic disorders and
cancers. (A related blotting technique, called Northern blot, can be used to determine
whether a cloned gene is transcriptionally active in a given cell or tissue type by probing for
the presence of mRNA that is complementary to a cloned gene. This is done by extracting
mRNA from one or several cell or tissue type. Following this somewhat perverse logic,
another procedure involving proteins bound to a filter is known as a Western blot.)
GENE PROBES
Success in isolating the desired recombinant clones dependents on the availability of a
suitable probe. Gene-specific probes are obtained in several ways. Frequently they are
constructed with cDNA clones. If the gene of interest is expressed in a specific tissue or cell
type, its mRNA is relatively abundant. For example, reticulocyte mRNA may be enriched in
globin mRNA, and pancreatic cells in insulin mRNA. Although mRNA is not available in
sufficient quantity to serve as a probe, the desired species can be converted into cDNA by
reverse transcription. The cDNA copies are purified, spliced into appropriate vectors, and
cloned to provide adequate amounts of the required probe.
Probes also can be generated if the gene codes for a protein of known amino acid sequence.
Oligonucleotides, about 20 nucleotides or longer, that code for a characteristic amino acid
sequence are synthesized. These often are satisfactory probes since they will specifically
bind to the gene segment coding for the desired protein.

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Sometimes previously cloned genes or portions of genes may be used as probes. This
approach is effective when there is a reasonable amount of similarity between the nucleotide
sequences of two genes.
After construction, the probe is labeled to aid detection. Often 32P is added to both DNA
strands so that the radioactive strands can be located with autoradiography. Nonradioactively
labeled probes may also be used.
CLONING VECTORS
Fragments of DNA produced by restriction enzyme digestion cannot directly enter bacterial
cells for cloning; when a DNA fragment is joined to a vector, however, it can gain entry to a
host cell, where it can be replicated or cloned into many copies. Vectors are, in essence,
carrier DNA molecules. To serve as a vector, a DNA molecule must have several properties:
1. It must be able to independently replicate itself and the DNA segments it carries.
2. It should contain a number of restriction enzyme cleavage sites that are present only
once in the vector. This site is cleaved with a restriction enzyme and used to insert
DNA fragments cut with the same enzyme.
3. It should 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 vectors.
4. It should be easy to recover from the host cell.
There is large number of vectors (plasmids, bacteriophages and other viruses, cosmids, and
artificial chromosomes) currently in use, which permits the cloning of DNA fragments over a
wide size range.
Plasmid as vector: Plasmids are
naturally occurring,
extrachromosomal, and are
mostly small, circular double
stranded DNA molecules that can
exist independently of host
chromosomes and replicate
autonomously, present in many
bacteria, also present in some
yeasts and other fungi. They have
their own replication origins and
stably inherited. Plasmids have a
wide range of structures: they
may be composed of DNA or
RNA, they may be double- or
single -stranded, and they may be
circular or linear. The smallest
bacterial plasmids are about
1.5kb and the largest are greater
than 1500kb. The vast majority

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are circular. However, several very large linear DNA plasmids, up to 500kb long, have been
found in species of Streptomyces and Nocardia.
Plasmids have relatively few genes, generally less than 30. Their genetic information is not
essential to the host, and bacteria that lack them usually function normally. Single copy
plasmids produce only one copy per host cell. Multicopy plasmids may be present at
concentrations of 40 or more per cell.
Plasmids may be classified in terms of their mode of existence and spread. An episome is a
plasmid that can exist either with or without being integrated into the hosts chromosome.
Some plasmids, conjugative plasmids, have genes for pili and can transfer copies of
themselves to other bacteria during conjugation.
Plasmids were the first cloning vectors. They are easy to isolate and purify, and they can be
reintroduced into a bacterium by transformation. Plasmids often bear antibiotic resistant
genes, which are used to select their bacterial hosts. A recombinant plasmid containing
foreign DNA often is called a chimera (after the Greek mythological monster that had the
head of a lion, the tail of a dragon, and the body of a goat.). One of the most widely used
plasmids is pBR322.
Plasmid pBR322 has both resistant genes for ampicillin and tetracycline and many restriction
sites. Several of these restriction sites occur only once on the plasmid and are
located within an antibiotic resistant gene. This arrangement aids detection of recombinant
plasmids after transformation. For example, if foreign DNA is inserted into the ampicillin
resistant gene, the plasmid will no longer confer resistant to ampicillin.
Fig: The pBR322 Plasmid. A map of the E. coli plasmid pBR322. The plasmid has resistance
genes for ampicillin (Apr) and tetracycline (Tett).
Many genetically engineered plasmid vectors are now available, and they offer a number of
useful features that make it easier to identify host cells carrying a plasmid with an inserted
DNA fragment. One such plasmid is pUC18. As a vector, this plasmid has several useful
properties:
1. The plasmid is small, allowing it to carry relatively large DNA inserts.
2. In a host cell, pUC18 can replicate to form about 500 copies per cell, producing many
clones, or copies, of inserted DNA fragments.
3. A large number of restriction enzyme sites have been engineered into pUC18, and
these are conveniently clustered in
one region, called a multiple-cloning
polylinker site.
4. The pUC18 plasmid has a selection
system that allows recombinant
plasmids to be identified.The pUC18
plasmid carries a fragment of the
bacterial lacZ gene, and the polylinker
is inserted into this fragment. Through
action of the lacZ gene, bacterial host
cells carrying pUC18 will produce
blue colonies when grown on medium

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 containing a compound called X-gal. When a DNA fragment is inserted into the
polylinker site, the lacZ gene is inactivated, and a bacterial cell carrying pUC18 with
an inserted DNA fragment will form white colonies, making them easy to identify.

Fig: The plasmid pUC18 offers several advantages as a vector for cloning.
The rDNA molecules are cloned by inserting them into bacteria, using transformation or
phage injection each strain reproduces to yield a population containing a single type of
recombinant molecule. The overall process is outlined in the following figure.
Cellular genome
Treatment with restriction enzymes
Mixture of DNA fragments
Agarose gel electrophoresis
Southern blotting
Band containing desired fragment
Extraction of DNA
Electrophoresis on different gel
Isolated DNA fragment
Anneal with plasmid or phage vector
DNA ligase treatment
Recombinant Vector
Transformed bacterial host
Culture bacteria
Isolated recombinant clones
Fig: Cloning Cellular DNA
Fragments. The preparation of
recombinant clone from
previously isolated DNA
fragments.
Phage vector
Both single- and double-stranded
phage vectors have been
employed in recombinant DNA
technology. For example, lambda
phage derivatives are very useful
for cloning and can carry
fragments about 40 kb in length.
The genes for lysogeny and
integration often are
nonfunctional and may be deleted
to make room for the foreign
DNA. The modified phage
genome also contains restriction
sequences in areas that will not
disrupt replication. After inserting
the foreign DNA into modified

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lambda vector chromosome, the recombinant phage genome is packaged into viral capsids
and can be used to infect host E. coli cells. These vectors are often used to generate genomic
libraries.E. coli also can be directly transformed with recombinant lambda DNA and produce
phages. However, this approach is less efficient than the use of complete phage particles.
The process is sometimes called transfection.

Cosmids
Cosmids are plasmids that contain lambda phage cos sites, necessary for packing Phage
DNA into phage protein coats and can be packaged into phage capsids. The lambda genome
contains a cos site at each end. When the genome is to be packaged in a capsid, it is cleaved
at one cos site and the linear DNA is inserted into the capsid until the second cos site has
entered. Thus any DNA between the cos sites is packaged. Cosmids typically contain several
restriction sites and antibiotic resistant genes. They can be packaged in lambda capsids for
efficient injection into bacteria, but they also can exist as plasmids within a bacterial host. As
much as 50kb of DNA can be carried in this way.

Bacterial Artificial Chromosome: The mapping and analysis of large complex eukaryotic
genomes requires cloning vectors that can accommodate very large DNA fragments. In

addition, since some human genes range from 1000kb to over 2000kb, vectors with large
cloning capacities are useful in studying the organization of these genes. Recently, a number
of vectors that use bacterial host cells have been developed.
One of these vectors is based on the fertility plasmid (F factor) of bacteria and is called
bacterial artificial chromosome (BAC). Because F factors can carry fragments of the
bacterial chromosome up to 1Mb in length, they have been engineered to act as vectors for
eukaryotic DNA and can carry inserts of about 300kb. BAC vectors carry the F factor genes
for replication and copy number, and incorporate an antibiotic resistance marker and
restriction enzyme sites for inserting foreign DNA to be cloned. In addition, the cloning site
is flanked by promoter sites that can be used to generate RNA molecules for the expression
of the cloned gene, for use as probes in chromosome walking, and for DNA sequencing of
the cloned insert.

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Shuttle Vectors
A shuttle vector is a cloning vector that allows it to replicate in two or more host organisms.
Shuttle vectors are used for experiments in which recombinant DNA is to be introduced into
organisms other than E coli. For example, the yeast-E.coli shuttle vector YEp24, which can
be introduced into yeast or E. coli cells.
Like E coli cloning vectors, YEp24 has an ori sequence that allows it to replicate in E. coli
and has dominant selectable markers that confer ampicillin resistance and tetracycline
resistance upon E.coli cells that contain this vector. YEp24 also contains the selectable
marker URA3 (a wild type yeast gene for an enzyme required for uracil biosysthesis). This
marker enable yeast ura3 mutant host cells containing YEp24 to be identified. That is, if
YEp24 transforms a yeast cell carrying a ura3 mutation, the yeast cells phenotype would be
changed from uracil-requiring to uracil- independent by the presence of the URA3 gene in
the YEp24 vector. YEp24 also carries a yeast-specific sequence, the two-micron circle (2u),
that allows it to replicate autonomously in a yeast cell. Thus, YEp24 is able to replicate in
both yeast and E.coli. Not all shuttle vectors have the ability to replicate in the nonbacterial
host. Those that do not will typically integrate into a host cells chromosome and be replicate
as that chromosome replicates.
EXPRESSION VECTORS
The study of a specific protein by recombinant DNA technology entails cloning the gene that
encodes the protein from desired organism, forming a suitable genetic construct such that
the gene can be expressed in a host organism, and introduction and maintenance of the
construct in the host to allow the production of an adequate yield of functional protein.
A cloned gene is not always expressed in the host cell without further modification of the
recombinant vector. To be transcribed, the recombinant gene must have a promoter that is
recognized by the host RNA polymerase. Translation of its mRNA depends on the presence
of leader sequences and mRNA modifications that allow proper ribosome binding. These are
quite different in eukaryotes and
prokaryotes, a prokaryotic leader must be
provided to synthesize eukaryotic proteins
in bacterium. Finally, introns in the
eukaryotic genes must be removed
because the prokaryotic host will not
excise them after trascription of mRNA; a
eukaryotic protein is not functional
without intron removal prior to translation.
Thus, before a specific RNA or protein
product can be produced in a particular
host cell, a suitable DNA construct must
be prepared. The expression system is
composed of an expression vector and a
specific host cell. Not only is the selection
of the ideal expression vector important,
Fig: Typical prokaryotic expression vector
but the choice of the appropriate host can

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affect production efficiency considerably.
Expression vectors usually consist of small, circular plasmids specifically designed with
several key features that allow a foreign gene inserted into the plasmid to be expressed in the
host cell. Important elements of the plasmid include:
1. an origin of replication, which allows the plasmid to be replicated in the host,
2. a selectable genetic
marker, which
allows cells bearing
the plasmid to
preferentially grow
on a specifically
composed medium,
3. transcription and
translation signals
recognized by the
host cell, and
4. a suitable unique
restriction site
located appropriately
with regard to the
transcription and
translation signals,
where the plasmid
can be cleaved and
the foreign DNA can
be introduced.
The most widely used
expression vectors for
E.coli are derived from
that of the CoIE1
plasmid and have copy
numbers of between 10
and 100. The useful
properties of a high
gene dosage have been
used advantageously
through the construction
of temperature sensitive
copy number control
systems in which the
copy number can be
raised dramatically by
increasing the

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temperature. A typical prokaryotic expression vector is represented in the following
diagram:
These vectors are often derivatives of plasmid pBR322 and contain the necessary
transcription and translation start signals. Some expression vectors contain portions of the
lac operon and can effectively regulate the expression of the cloned genes in the same
manner as the operon.
Somatostatin, the 14-residue hypothalamic polypeptide hormone that helps regulate
human growth, provides an example of useful cloning and protein production. Besides the
42 bases coding for somatostatin, the polynucleotide contain a codon for methionine at
the 5-prime end (the N-terminal end of the polypeptide) and two stop codons at the
opposite end. To aid insertion into the plasmid vector, the 5-prime ends of the synthetic
gene were extended to form single stranded sticky ends complementary to those formed
by EcoRI and BamHI restriction enzymes. A modified pBR322 plasmid was cut with
both EcoRI and BamHI to remove a part of the plasmid DNA. The synthetic gene was
then spliced into the vector by taking advantage of its cohesive ends. Finally, a fragment
containing the initial part of the lac operon (including the promoter, operator, ribosome
binding site, and much of the beta-galactosidase gene) was inserted next to the
somatostatin gene. The plasmid now contained the somatostatin gene fused in the proper
orientation to the remaining portion of the beta-galactosidase gene.
After introduction of the chiremic plasmid into E.coli, the somatostatin gene was
transcribed with the beta-galactosidase gene fragment to generate an mRNA having both
messages. Tranaslation foremed a protein consisting of the total hormone polypeptide
attached to the beta-galactosidase fragment by a methionine residue. Cyanogen bromide
cleaves peptide bonds at methinine residues and released the hormone. Once free, the
polypeptide was able to fold properly become active. A similar approach was used to
manufacture human insulin.
GENOMIC LIBRARIES
Because each cloned DNA segment is relatively small, many separate clones are required
to include even a small portion of an organisms genome. A set of DNA clones derived
from a single individual represents a library. Cloned libraries can represent an entire
genome, a single chromosome, or a set of genes that are actively transcribed in a single
cell type.
Ideally, a genomic library is a collection of clones that contains at least one copy of all the
sequences represented in the genome. One approach to obtaining a clone of a gene is to
isolate it from a genomic library through the use of a specific probe.
There are three ways to produce genomic libraries:
1. Genomic DNA is completely digested by a restriction enzyme, and the resulting DNA
fragments are then cloned in a cloning vector. This technique does have a drawback. If
the specific gene the researchers want to study contains restriction sites for the
enzyme, the gene will be split into two or more fragments when the DNA is digested
by the restriction enzyme. In this case, the gene would then be cloned in two or more
fragments. Another drawback is that the average size of the fragment produced by

14
 digestion of eukaryotic DNA with restriction enzymes is relatively small. Thus, an
entire library would need to contain a very large number of recombinant DNA
molecules, and screening for the specific gene would be very laborious.
2. The problems of genes split into fragments and the large number of recombinant DNA
molecules can be minimized by cloning longer DNA fragments. Longer DNA
fragments can be generated by mechanically shearing high-molecular weight (usually
100 to 150 kb) DNA. For example, the passage of the syringe needle will produce a
population of overlapping DNA fragments. However, since the ends of the resulting
DNA fragments have not been generated by cutting with restriction enzymes,
additional enzymatic manipulations are necessary to add appropriate ends to the
molecules for insertion into vector cloning sites.
3. Another approach for producing DNA fragments of appropriate size for constructing a
genomic library is to perform a partial digestion of the DNA with restriction enzymes
that recognize frequently occurring four-base pair recognition sequences. Partial
digestion means that only a portion of the available restriction sites is actually cut with
the enzyme. This achieved by limiting the amount of the enzyme used and/ or the
time of incubation with the DNA. The ideal result of partial digestion is a population
of overlapping fragments representing the entire genome. Sucrose gradient
centrifugation or agarose gel electrophoresis is then used to collect fragments of the
desired size for cloning. Those fragments can be cloned directly since the ends of the
fragments were produced by restriction enzyme digestion.
The recombinant DNA molecules produced by this method are used to transform E.coli.In
the case of plasmid and cosmid libraries, the transformants are plated on selective
medium to clone the sequences. Each colony that is produced almost always represents a
different cloned DNA sequences since each bacterium that gave rise to a colony most
likely contained a different recombinant DNA molecule.
The aim of this method to produce a library of recombinant molecules that is as complete
as possible. However, not all sequences of the eukaryotic genome are equally represented
in such a library, for example, if the restriction sites in a particular region are very far
apart or extremely close together, the chances of obtaining a fragment of clonable size are
small.
Lastly, the probability of having at least one copy of any DNA sequence represented in
the genomic library can be calculated from the formula:
In(1-P)
N= ------------ Where N is the necessary number of recombinant DNA
. In( 1-f) molecules , P is the probability desired, and f is the fractional
proportion of the genome in a single recombinant DNA
molecule. Suppose that we wish to prepare a library of human genome using a lambda
phage vector. The human genome contains 3.0 X 106kb of DNA, and the average size of a
cloned insert that can be carried by the vector is 17kb; therefore, about 8.1 X 10 5 phages
would be required to establish a library housing all sequences. If we had selected a

15
plasmid vector with a capacity of only 5kb, several million clones would be needed to
create the library.
CHROMOSOME LIBRARIES: The genome size of many organisms, including
humans, is so large that many thousands of clones are needed to represent the entire
genome. This makes searching the library for a gene of interest very time consuming. One
approach for reducing the searching time of large genomes is to make libraries of
individual chromosomes in the genome. In humans, this gives 24 different libraries, one
each for 22 autosomes, the X and the Y. Then, if a gene has been localized to a
chromosome by genetic means, researchers can restrict their attention to the library of that
chromosome when they search for its DNA sequence.
Individual chromosomes of an organism can be separated if their morphologies and size
are distinct enough, as is the case for human chromosomes. One procedure currently used
to isolate large chromosomes individually is flow cytometry. In this procedure,
chromosomes from cells in the mitotic phase of the cell cycle are stained with two
fluorescent dyes, one that binds to A=T pairs, the other to G=C pairs. The stained
chromosomes flow passed a laser beam connected to light detector. This system sorts and
fractionates the chromosomes based on their differences in dye binding and resulting light
scattering. Approximately 100 chromosomes can be sorted and isolated per second. .
Once the chromosomes have been fractioned, a library of each chromosome type can be
made by cutting the chromosomal DNA with restriction enzymes and inserting the
fragments into cloning vector. As a result of the application of these procedures, libraries
of DNA prepared from all human chromosomes are now available to researches.
cDNA LIBRARIES
DNA copies, called complementary DNA (cDNA), can be made from mRNA molecules
isolated from cells. These cDNA molecules can be cloned. Thus, if a specific mRNA
molecule can be isolated, the corresponding cDNA can be made and cloned. The analysis
of that cloned cDNA molecule can then provide information about the gene that encoded
the mRNA. More typically, the entire mRNA population of a cell is isolated and a
corresponding set of cDNA molecules is made and inserted into a cloning vector to
produce a cDNA library. Since a cDNA library reflects the gene activity of the cell type
at the time the mRNAs are isolated, the construction and analysis of cDNA libraries is
useful for comparing gene activities in different cell types of the same organism, because
there would be similarities and differences in the clones represented in the cDNA libraries
of each cell type.
Recognize that the clones in the cDNA library represented the mature mRNAs found in
the cell. In eukaryotes, mature mRNAs are processed molecules having no introns, so the
sequences obtained are not equivalent to gene clones. So gene clones are much more
informative than can cDNA clone, for example, on the presence and arrangement of
introns, and on the regulatory sequences associated with gene.
POLYMERASE CHAIN REACTION (PCR)
The generation of large numbers of identical copies of DNA by the construction and
cloning of recombinant DNA molecules was possible in the 1970s.Indeed, recombinant
DNA techniques revolutionized molecular genetics by making it possible to analyze

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genes and their functions in new ways. However, the cloning of DNA is time consuming,
involving the insertion of DNA into
cloning vectors and typically the
screening of libraries to detect
specific DNA sequences. In the mid-
1980s, the polymerase chain
reaction (PCR) was developed and
this has resulted it yet a new
revolution in the way genes may be
analyzed.
PCR is a rapid cell-free method for
producing an extremely large
number of copies of a specific DNA
sequence from a DNA mixture
without having to clone it, a process
called amplification. That is, PCR
permits the selective amplification of
DNA sequences. Kary Mullis, who
developed PCR, was awarded the
Nobel Prize in 1993.
The starting point for PCR is the
DNA mixture containing the DNA
sequence to be amplified, a pair of
oilgonucleotide primers that flank
that DNA sequence of interest.the
primers are made synthetically, so
some information must be available
about the specific DNA sequence of
interest so that it can be amplified. In
brief, the PCR procedure is as
follows:
1. Denaturing the DNA to single
strands by incubating at 94oC.
Cool (to 37-550C, depending
on how well the base
sequences of the primers
matches the base sequence of
the DNA) and anneal the
specific pair of primers
(primer A and primer B) that
flank the target DNA
sequence.
2. Extend the primers with DNA polymerase. For this a specific heat- resistant DNA
polymerase called Taq ( tack) polymerase is used.

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 3. Repeat the heating cycle to denature the DNA to single strands and cooling to
anneal new primers.
4. Repeat the primer extension with Taq DNA polymerase. In each of the two double
stranded molecules produced, one strand of unit length; that is, it is the length of
the DNA between the 5-prime end of primer A and the 5-prime end of the primer
B- the length of target DNA. The other strand of both molecules is longer than unit
length.
5. Repeat the denaturation of DNA and anneal the new primers.
6. Repeat the primer extension with Taq polymerase. This produces unit-length,
doublestranded DNA. Note that it took three cycles to produce the two molecules
of target-length DNA. Repeated denaturation, annealing, extension cycles results in
geometric increase of the unit- length DNA. Amplification of longer-than- unit-
length DNA occurs simultaneously, but only in a linear fashion.

Using PCR, the amount of new DNA generated increases geometrically. Starting with
one molecule of DNA, one cycle of PCR produces two molecules, two cycles
produces four molecules, and three cycles produces eight molecules, two of which are
the target DNA. A further ten cycles produces 1,024 copies ( 2 10) of the target DNA
and in 20 cycles there will be 1,048,576 copies ( 2 30) of the target DNA! The
procedure is rapid, each cycle taking only a few minutes using a termal cycler, a
machine that automatically cycles through the temperature changes in a programmed
way.
Only the disadvantage of the PCR procedure is that Taq polymerase does not have
proof reading properties, so that errors are introduced into the DNA copies at low
frequencies. Of course, should an error be introduced in an early cycle of PCR, then
all subsequent copies made from that DNA would have that error. PCR also
susceptible to contamination. That is if the reaction mixture becomes contaminated
with other DNA to which the primers can bind, the n that DNA can also be amplified
as well the target DNA.
There are many applications for PCR, including amplifying DNA for cloning,
amplifying DNA from genomic DNA preparations for sequencing without cloning,
mapping DNA segments, disease diagnosis, sex determination of embryos, forensics,
studies of molecular evolution, etc. In each case, some DNA sequence information
must be available so that appropriate pairs of primers can be synthesized.
APPLICATIONS OF GENETIC ENGINEERING
Genetic engineering and biotechnology will contribute in the future medicine, industry,
and agriculture, as well as to basic research. Some practical applications are as follows:
Medical applications: Certainly the production of medically useful proteins such as
somatostatin, insulin, human growth hormone, and interferon are of great practical
importance. This is particularly true of substances that previously only could be
obtained from human tissues. For example, in the past, human growth hormone for the

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treatment of pituitary dwarfism was extracted from pituitaries obtained during
autopsies and was available only in limited amounts.
Interleukin-2 (a protein that helps regulate the immune response) and blood clotting
factor VIII have recently been cloned, and undoubtedly other important peptides and
proteins will be produced in future.
It also be possible to use genetically engineered plants (transgenic plants) to produce
oral vaccines. A recombinant hepatitis B vaccine is already commercially available.
Probes are now being used in the diagnosis of infectious diseases. An individual could
be screened for mutant genes with probes and hybridization techniques (even before
birth when used together with amniocentesis).
A type of genetic surgery called somatic cell gene therapy may be possible for
artificial individuals. For example, cells of individual with a genetic disease could be
removed, cultured and transformed with cloned DNA containing a normal copy of yhe
defective gene(s). These could then be reintroduced into the individual; if they become
established, the expression of normal genes might cure patients. Recently so many
techniques have been developed by different researchers (including the virosome
model, the most efficient technique of gene delivery, developed by Dr. D.P.Sarkar of
Delhi University) to transfer correct genes to the patient. After successful experiments
with rats, human clinical trials are in process for transferring the normal CF gene (an
autosomal recessive mutation) to cystic fibrosis patients. Encouraging results have
been obtained in the treatment of leukemia, lymphomas, and rheumatoid arthritis.
However, many scientific, ethical, and legal questions will have to be addressed before
the routine implantation of gene therapy.
Industrial applications: Industrial applications of recombinant DNA technology
include manufacturing protein products by using bacteria, fungi, and cultured
mammalian cells as factories; strain improvement for existing bioprocesses; and the
development of new strains for additional bioprocesses. The pharmaceutical industry
is already producing several medically important polypeptides using this technology.
In addition, there is interest in making expensive, industrially important enzymes with
recombinant bacteria. Bacteria that metabolize petroleum and other toxic materials
have been developed. These bacteria can be constructed by assembling the necessary
catabolic genes on a single plasmid and then transforming the appropriate organism.
Agricultural applications: It is also possible to bypass the traditional methods of
selective breeding and directly transfer desirable traits to agriculturally important
plants and animals. Potential exists for increasing growth rates and overall protein
yields of farm animals. The growth hormone gene has already been transferred from
rats to mice, and both in vitro fertilization and embryo implantation methods are fairly
well developed. Recently, recombinant bovine growth hormone has been used to
increase milk production by at least 10%. Perhaps farm animals disease resistance
and tolerance to environmental extremes also can be improved.
Cloned genes can be inserted into plant as well as animal cells. Presently a popular
way to insert genes into into plants is with a recombinant Ti plasmid (tumor-inducing
plasmid) obtained from the bacterium Agrobacterium tumefaciens.

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 Much effort has been developed to the transfer of the nitrogen fixing abilities of
bacteria associated with legumes to other crop plants. Attempts at making plants
resistant to environmental stress have been more successful.
Analysis of biological processes and basic research: One of the fundamental and
widespread applications of recombinant DNA and PCR methodologies is in basic
research to explore biological functions. In genetics, researchers are investigating such
things as the functional organization of genes and the regulation of gene expression. In
developmental biology, key regulatory genes and target genes responsible for
developmental events are being discovered and analyzed, and gene changes associated
with aging and cancer are being investigated. In evolutionary biology, DNA sequence
analysis is adding new information about the evolutionary relationships between
organisms.
DNA Fingerprinting: The Ultimate Identification Test:
Everyone is familiar with the use of fingerprints in forensic science. The principle is
that no two individuals have the same fingerprints so that fingerprint left at the scene
of a crime is important evidence in a criminal investigation. Similarly, no two human
individuals (except identical twins) have exactly the same genome, base pair for base
pair, and this has led to the development of DNA techniques for use in forensic
science, in paternity and maternity testing, and elsewhere.
The technique of DNA fingerprinting, also called DNA typing or DNA profiling,
relies on developments from recombinant DNA technology allows an examination of
each individuals unique genetic blueprint-DNA. The technique was discovered in
England by Alec Jeffreys. It is based on the fact that the DNA of each individual is
interrupted by a series of identical DNA sequences, called repetitive DNA or tandem
repeats .The pattern, length, and number of these repeats are unique for each
individual. Jeffreys developed a series of DNA probes, which are short pieces of DNA
that seek out any specific sequence they match, and base pair with that sequence. Such
molecular probes are used to detect the unique repetitive DNA patterns characteristic
of each individual. The procedure of DNA fingerprinting has the following steps:
1. DNA is purified from a small sample of blood, semen, or other DNA bearing
cells, and digested into smaller fragments with restriction endonucleases.
2. The fragments are separated by agarose gel electrophoresis.
3. The separated fragments are transferred to a nylon membrane by the technique of
Southern blotting.
4. The DNA probes labeled with radioactive material are added to a solution
containing membrane.
5. Wherever the probes fit a band containing repetitive DNA sequences, they attach.
6. The X-ray film is pressed against the nylon filter and exposed at bands carrying
the radioactive probes attached to the fragments.
7. The pattern of bands obtained on the film is 100 percent unique for each person,
except for identical twins who would have the same pattern.

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In forensic application the DNA fingerprinting technique involves a comparison between
the DNA fingerprint obtained from cells at a crime scene with a DNA fingerprint from
cells provided by the suspect. If the DNA pattern matches exactly, certain identification is
made. For paternity determination, DNA fingerprints of the mother, child and alleged
father are compared. In this case, one- half of the bands in the child come from the mother
and other half from the father. All the paternal bands in childs DNA fingerprint must
match with the alleged father for positive paternity identification.
In India, DNA fingerprinting tests are carried out at the Centre for Cell and Molecular
Biology (CCMB), Hyderabad. For this purpose, a test with the BKM-DNA probe (=
banded krait minor satellite DNA) earlier used for identification of sex chromosome (by
Dr. Lalji Singh) has been found to cost of tests used in Europe and U.S.A. Paternity
dispute cases are much more common in India and most of them are referred to CCMB for
DNA evidence. The first such test on DNA fingerprinting was used in June 1989 to settle a
drawn-out paternity case in Madras.

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