SUBJECT CODE:-CONCEPTS OF BIOTECHNOLOGY

TOPIC: -ENZYMES USED IN GENETIC ENGINEERING

SUBJECT - BTC012

SUBMITTEDTO: - SUBMITTED BY:-

M R.HARSH ROLL NO.R108B30

(LECT. IN BIOTECH) REG.NO.1040070105

B.TECHBIOTECH

RD
SEM
3
 PREFACE

Genetic engineering is a new technology that combines genes from totally unrelated
species, in combinations not possible using conventional breeding methods. Genes from
an animal, say, a fish, can be put into a plant, a strawberry for instance.. So different
type of enzymes are used in genetic engineering

In fact this is an actual example of an attempt to "improve" strawberry plants. The fish
gene is supposed to make the strawberries more resistant to frost by causing the
strawberry plant to produce a form of antifreeze which the fish normally produces to
endure cold ocean conditions.

The primary goal of this term parer has been to provide useful information about genetic
engineering in a simple and illustrative language . The organization of the material Is
largely traditional and the order in which the content arranged, is a matter of personal
choice. I hope you will enjoy reading.

 Thank you
 ACKNOWLEDGEMENT

I would like to express my gratitude to all those who gave me the possibility to complete
this term paper. I want to thank the Department of BIOTECHNOLOGY of LOVELY
PROFESSIONAL UNIVERSITY for giving me permission to commence this Term
paper, to do the necessary research work and to use departmental data. I have
furthermore to thank to Mr.Harsh, Lect.in concepts in biotechnology, who gave and
confirmed this permission and encouraged me to go ahead with my term paper.

I am deeply indebted to my supervisor Lect. Harsh whose help, stimulating suggestions

and encouragement helped me in all the time of research for and writing of this term
paper.

Thank you
 CONTENT

• Introduction

• W hat is genetic engineering

• Principle of genetic engineering

• Enzymes used in genetic engineering

• Applications of genetic engineering

• References
 INTRODUCTION

There are many enzymes used in molecular cloning and genetic engineering. DNA
polymerase I and kle
now fragment are used for filling the gap reaction and replication.
DNA polymerase is used during dideoxy DNA sequencing for extending the growing
complemetary chain of DNA . klenow fragment the larger fragment of DNA polymeraseI
and devoid of 5’,3’ exonuclease activity issued for replication of the complementary
chain during olinucleotide site directed mutagenesis, reverse transcriptase ,capable of
synthesizing DNA from RNA has become important for synthesizing complementary
DNA from Mrna for making Cdna library. Polynuleotide kinase tranfers-y phosphoryl
group from ATP with the phosphoryl group present at the 5’end of the DNA. Terminal
deoxynucleotidyl transferase is capable of trasnferring deoxyribonucleotidyl onto the 3’
end of DNA strands in a template independent reaction using deoxynucleoside
triphosphate. It is used for labelling 3’ end of DNA and labelled DNA is further
sequencing purpose by the method based on chemical modification method of maxam &
gilbert. Alkaline phosphate remove phosphate group from phosphoryl esters. In
molecular biology it is used to dephosphorylate DNA at the 5; end . S1 nuclease
degrades single strand nucliec acid . S1 nuclease is used to remove single stranded tail
from double stranded DNA to produce blunt ends. DNA LIGASE is used for end to end
joining of DNA for cloning DNA fragment in the vector.
WHAT IS GENETIC ENGINEERING

Genetic engineering is the alteration of genetic material by direct intervention in genetic

processes with the purpose of producing new substances or improving functions of
existing organisms, it offers the possibility of cures for diseases and countless material
improvements to daily life. Hopes for the benefits of genetic engineering are symbolized
by the Human Genome Project, a vast international effort to categorize all the genes in
the human species.

Genetic engineering
, recombinant DNAtechnology, genetic modification/manipulation
(GM) and gene splicingare terms that apply to the direct manipulation of
organism
an 's
genes. Genetic engineering is different from traditional
breeding, where the organism's
genes are manipulated indirectly; genetic engineering uses the techniques
molecular
of
cloning and transformationto alter the structure and characteristics of genes directly.
Genetic engineering techniques have found some successes in numerous applications.
Some examples are in improving crop technology, the manufacture of synthetic human
insulin through the use of modified
bacteria, the manufacture oferythropoietin in
hamsterovary cells, and the production of new types of experimental mice such as the
oncomouse(cancer mouse) for research
.

Principles of Genetic Engineering

Just as DNA is at the core of studies in genetics,

recombinant DNA(rDNA)—that is,
DNA that has been genetically altered through a process known
geneassplicing— is the
focal point of genetic engineering. In gene
splicing, a DNA strand is cut in half
lengthwise and joined with a strand from another organism or perhaps even another
species. Use of gene splicing makes possible two other highly significant techniques.
Gene transfer, or incorporation of new DNA into an organism's cells, usually is carried
out with the help of microorganism
a that serves as a vector, or carrier. Gene therapy is
the introduction of normal or genetically altered genes to cells, generally to replace
defective genes involved in genetic disorders.

DNA also can be cut into shorter fragments through the use of restriction enzymes. (An
enzyme is a type of protein that speeds up chemical reactions.) The ends of these
fragments have anaffinity for complementaryends on other DNA fragments and will
seek those out in the target DNA. By looking at the size of the fragment created by a
restriction enzyme, investigators can determine whether the gene has the proper genetic
code. This technique has been used to analyze genetic structures
fetalincells and to
diagnose certain blood disorders, suchsickle
as cell anemia
.
Gene Transfer

Suppose that a particular base-pair sequence carries the instruction "make insulin"; if a
way could be found to insert that base sequence into the DNA of bacteria, for example,
those bacteria would be capable of manufacturing
insulin. This, in turn, would greatly
improve the lives of people with type 1 diabetes, who depend on insulin shots to aid their
bodies in processing blood sugar.

The first person tosurmount these obstacles was the American biochemist Paul Berg
(1926-), often referred to as the "father of genetic engineering." In 1973 Berg developed
a method for joining the DNA from two different organisms, a monkey virus known as
SV40 and a virus calledlambda phage.Then, later that year, the American biochemists
Stanley Cohen (1922-) at Stanford University, and Herbert Boyer (1936-) at the
University of California at San Francisco discovered an enzyme that greatly increased
the efficiency of the Berg procedure. The gene-transfer technique developed by Berg,
Boyer, and Cohen formed the basis for much of the ensuing progress in genetic
engineering.
ENZYMES USED IN GENETIC ENGINEERING

The ability to manipulate DNA in vitro (outside the cell) depends entirely on the availability of purified
enzymes that can cleave, modify and join the DNA molecule in specific ways.
At present, no purely chemical method can achieve the ability to manipulate the DNA in vitro in a
predictable way. Only enzymes are able to carry out the function of manipulating the DNA. Each
enzyme has a vital role to play in the process of genetic engineering.

• DNA LIGASE

In molecular biology
, DNA ligaseis a special type ofligase that can link together two
DNA strands that
have single-strand breaks (a break in both complementary strands of DNA). The alternative, a double-
strand break, is fixed by a different type of DNA ligase using
complementary
the strand
as a template
but still requires DNA ligase to create the final
phosphodiester bond
to fully repair the DNA.

DNA ligasehas applications in both

DNA repairand DNA replication
. In addition, DNA ligase has
extensive use in molecular biology laboratoriesGenetic
for recombination
experiment

DNA Ligase- Recombinant DNA experiments require the joining of two different DNA segments or
fragments in vitro.

The cohesive ends generated by some RE will anneal (join) themselves by forming hydrogen bonds.

But the segments annealed thus are weak and do not withstand experimental conditions.

To get a stable joining, the DNA should be joined by using an enzyme called ligase.

DNA ligase joins the DNA molecule covalently by catalysing the formation of phosphodiester bonds
between adjacent nucleotides.
DNA ligase isolated from E. coli and. T 4 bacteriophage is widely used in molecular biology
experiments.

These ligases more or less catalyse the reaction in the same way, and differ only in requirements of
cofactor. T4 ligase requires ATP as cofactor and E coli ligase requires NADP as cofactor.

The cofactor is first split (A TP à AMP + 2Pi) and then AMP binds to the enzyme to form the enzyme-
AMP complex.

This complex then binds to the nick or break (with 5' -PO4 and 3' -OH) and makes a covalent bond in
the phosphodiester chain. The ligase reaction is carried out at 4°C for better results.

Ligase mechanism

The mechanism of DNA ligase is to form covalent

two phosphodiester bonds
between3' hydroxyl ends
of onenucleotidewith the5' phosphate endof another. ATP is required for the ligase reaction.

Ligase will also work with

blunt ends, although higher enzyme concentrations and different reaction
conditions are required.

• NUCLEASE

A nucleaseis an enzymecapable of cleaving the

phosphodiester bonds
between the nucleotide subunits
of nucleic acids.Older papers may use terms such as "polynucleotidase" or "nucleodepolymerase".

In 1960s, scientistsStewart Linnand Werner Arberisolated examples of the two types of enzymes
responsible for phage growth restrictionEscherichia
in coli(E. coli) bacteria. One of these enzymes
added amethyl groupto the DNA, generating
methylated DNA
, while the other cleaved
unmethylated
DNA at a wide variety of locations along the length of the molecule. The first type of enzyme was
called a "m ethylase" and the other arestriction
" nuclease
"..

HindIIenzyme always cuts directly in the center of this sequence. Wherever this particular sequence
of six base pairs occurs unmodified in a DNA molecule, HindII will cleave
DNAbothbackbones
between the 3rd and 4th base pairs of the sequence. Moreover, HindII will only cleave a DNA molecule
at this particular site. For this reason, this specific base sequence is known recognition
as the "
sequence" for HindII.

HindII is only one example of the class of enzymes known as restriction nucleases.
EcoRI comes from
Escherichia coliRY13 bacteria, while HindII comes from
Haemophilus influenzae
strain Rd. Nucleases
are further described by addition of the prefix "endo" or "exo" to the name: The
endonuclease
term " "
applies to nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of
the molecule. A nuclease that functions by removing nucleotides from the ends of the DNA molecule is
called anexonuclease.

Endonucleases and DNA fragments

A restriction endonuclease functions by "scanning" the length of a DNA molecule. Once it encounters
its particular specific recognition sequence, it will bond to the DNA molecule and makes one cut in
each of the twosugar-phosphate backbones
of the double helix. The positions of these two cuts, both in
relation to each other, and to the recognition sequence itself, are determined by the identity of the
restriction endonuclease used to cleave the molecule in the first place. Different endonucleases yield
different sets of cuts, but one endonuclease will always cut a particular base sequence the same way, no
matter what DNA molecule it is acting on. Once the cuts have been made, the DNA molecule will break
into fragments.

Endonucleases and sticky ends

Not all restriction endonucleases cut symmetrically and leave blunt ends like HindII .Many
endonucleases cleave the DNA backbones in positions that are not directly opposite each other. For
example, the nuclease EcoRI has the following recognition sequence:

Enzym
Source Recognition Sequence Cut
e

5'GAATTC 5'---G AATTC---3'

EcoRI Escherichia coli
3'CTTAAG 3'---CTTAA G---5'
W hen the enzyme encounters this sequence, it cleaves each backbone between the G and the closest A
base residues. Once the cuts have been made, the resulting fragments are held together only by the
relatively weak hydrogen bonds that hold the complementary bases to each other. The weakness of
these bonds allows the DNA fragments to separate from each other. Each resulting fragment has a
protruding 5' end composed of unpaired bases. Other enzymes create cuts in the DNA backbone which
result in protruding 3' ends. Protruding ends—both 3' and 5'—are sometimes sticky
called ends
" "
because they tend to bond with complementary sequences of bases..
Ligase enzyme is then used to join
the phosphate backbones of the two molecules.

• Nucleases:- DNase and RNase

Most of the time nucleases are the enemy of the molecular biologist who is trying to preserve the
integrity of RNA or DNA samples. However, deoxyribonucleases (DNases) and ribonucleases
(RNases) have certain indispensible roles in molecular biology labs.

Deoxyribonuclease cleaves
I double-stranded or single stranded DNA. Cleavage
preferentially occurs adjacent to pyrimidine (C or T) residues, and the enzyme is therefore an
endonuclease.

Major products are 5'-phosphorylated di, tri and tetranucleotides.

In the presence of magnesium ions, DNase I hydrolyzes each strand of duplex DNA independently,
generating random cleavages. In the presence of manganese ions, the enzyme cleaves both strands
of DNA at approximately the same site, producing blunt ends or fragments with 1-2 base
overhangs. DNase I does not cleave RNA, but crude preparations of the enzyme are contaminated
with RNase A; RNase-free DNase I is readily available.

Some of the common applications of DNase I are:

• Eliminating DNA (e.g. plasmid) from preparations of RNA.

• Analyzing DNA-protein interactions via DNase footprinting.
• Nicking DNA prior to
radiolabelingby nick translation.

Ribonuclease Ais an endoribonuclease that cleaves single-stranded RNA at the 3' end of
pyrimidine residues. It degrades the RNA into 3'-phosphorylated mononucleotides and
oligonucleotides.

Some of the major use of RNase A are:

• Eliminating or reducing RNA contamination in preparations of plasmid DNA.

• Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in
RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be
analyzed.

• Alkaline Phosphatase

Alkaline phosphataseremoves 5' phosphate groups from DNA and RNA.

It will also remove
phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH - hence the
name.

There are several sources of alkaline phosphatase that differ in how easily they can be inactivated:

• Bacterial alkaline phosphatase (BAP)

is the most active of the enzymes, but also the most
difficult to destroy at the end of the dephosphorylation reaction.
• Calf intestinal alkaline phosphatase (CIP)
is purified from bovine intestine. This is phosphatase
most widely used in molecular biology labs because, although less active than BAP, it can be
effectively destroyed by protease digestion or heat (75C for 10 minutes in the presence of 5
mM EDTA).
• Shrimp alkaline phosphatase
is derived from a cold-water shrimp and is promoted for being
readily destroyed by heat (65C for 15 minutes).

There are two primary uses for alkaline phosphatase in DNA manipulations:

• Removing 5' phosphates from plasmid and bacteriophage vectors

that have been cut with a
restriction enzyme. In subsequent ligation reactions, this treatment prevents self-ligation of the
 vector and thereby greatly facilitates
ligation of other DNA fragments into the vector (e.g.
subcloning).

• Removing 5' phosphates from fragments of DNA prior to labeling with radioactive phosphate
.
Polynucleotide kinase
is much more effective in phosphorylating DNA if the 5' phosphate has
previously been removed.

It is usually recommended that dephosphorylation of DNAs with blunt or 5'-recessed ends be conducted
using a higher concentration alkaline phosphatase or at higher temperatures than for DNAs with 5'
overhangs

• POLYNUCLEOTIDE KINASE

Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5'
end of either DNA or RNA.
It is a product of the T4 bacteriophage, and commercial preparations are
usually products of the cloned phage gene expressed in E. coli.

The enzymatic activity of PNK is utilized in two types of reactions:

• In the "forward reaction"

, PNK transfers the gamma phosphate from ATP to the 5' end of a
polynucleotide (DNA or RNA). The target nucleotide is lacking a 5' phosphate either because it
has beendephorphorylatedor has been synthesized chemically.

• In the "exchange reaction"

, target DNA or RNA that has a 5' phosphate is incubated with an
excess of ADP - in this setting, PNK will first transfer the phosphate from the nucleic acid onto
an ADP, forming ATP and leaving a dephosphorylated target. PNK will then perform a forward
reaction and transfer a phosphate from ATP onto the target nucleic acid.
PNK is inhibited by small amounts of ammonium ions, so ammonium acetate should not be used to
precipitate nucleic acids
prior to phosphorylation. Low conceptrations of phosphate ions, or NaCl
concentrations greater than about 50 mM, also inhibit this enzymes.

• RESTRICTION ENZYME

DNase which act on specific positions of sequences on the DNA are called as restriction
endonucleases. The sequences which are recognized by the restriction endonucleases or restriction
enzymes are called as recognition sequences or restriction sites.
These sequences are palindromic sequences( if read from right to left ,the sequence is same). Different
restriction enzymes present in different bacteria can recognize different or same restriction sites. But
they will cut at two different points within the restriction sites. Such restriction enzymes are called as
isoschizomers.

A restriction enzyme(or restrictionendonuclease) is an enzyme that cuts double-stranded or single

strandedDNA at specific recognition
nucleotidesequences known as
restriction sites
. Such enzymes,
found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against
invadingviruses. Inside a bacterial host, the restriction enzymes selectively cut
foreign
up DNA in a
process calledrestriction; host DNA ismethylatedby a modification enzyme methylase
(a ) to protect it
from the restriction enzyme’s activity.

Different restriction enzymes that recognize the same sequence are known
neoschizomers
as . These
often cleave in a different locales of the sequence; however, the specific type that cleaves in the same
location as the prototype is known asisoschizomer
an .

Type I
Type I restriction enzymes were the first to be identified and are characteristic of two different strains
(K-12 and B) ofE. coli. These enzymes cut at a site that differs, and is some distance (at least 1000 bp)
away, from their recognition site. The recognition site is asymmetrical and is composed of two portions
– one containing 3-4 nucleotides, and another containing 4-5 nucleotides – separated by a spacer of
about 6-8 nucleotides. Several enzyme cofactors, including
S-Adenosyl methionine(AdoMet),
hydrolyzed adenosine triphosphate ( and magnesium(Mg2+) ions, are required for their activity.
ATP)
Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for
restriction, HsdM is necessary for adding
methyl groups to host DNA (methyltransferase activity) and
HsdS is important for specificity of cut site recognition in addition to its methyltransferase activity

1. In genetic engineering, scientists use restriction enzymes to isolate a segment of DNA that contains
a gene of interest, for example, the gene regulating insulin production. 2. A plasmid extracted from its
bacteria and treated with the same restriction enzyme can hybridize with this fragment’s “sticky” ends
of complementary DNA. 3. The hybrid plasmid is reincorporated into the bacterial cell, where it
replicates as part of the cell’s DNA. 4. A large number of daughter cells can be cultured and their
gene products extracted for human use.

Typical type IIrestriction enzymes differ from type I restriction enzymes in several ways. They
are composed of only one subunit, their recognition sites are usually undivided and palindromic and 4-8
nucleotides in length, they recognize and cleave DNA at the same site, and they do not use ATP for
their activity – they usually require only2+Mg
as a cofactor. These are the most commonly available and
used restriction enzymes

Type II Restriction Enzymes - Blend End Cutters

- Blunt end cutters Type II
restriction enzymes of this class cut the DNA strands at same points on both the strands of DNA within
the recognition sequence.

The DNA strands generated are completely base paired. Such fragments are called as blunt ended or
flush ended fragments.

Type II Restriction Enzymes - Cohesive End Cutters

- Cohesive end cutter Type
II

restriction enzymes of this class cut the DNA stands at different points on both the strands of DNA
within the recognition sequence.

They generate a short single-stranded sequence at the end

This short single strand sequence is called as sticky or cohesive end. This cohesive end may contain 5
-PO 4 or 3 -OH, based upon the terminal molecule (5 -PO4 or 3 -OH).

These enzymes are further classified as 5end cutter (if 5 -PO 4 is present) or 3 -end cutter (if3' -OH is
present).

Type III

Type III restriction enzymes (e.g.

EcoP15) recognize two separate non-palindromic sequences that are
inversely oriented. They cut DNA about 20-30 base pairs after the recognition site. These enzymes
contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA
methylation and restriction, respectively.

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However, type II restriction enzymes take the help of another enzyme called methylase, and methylate the
DNA. Then RE clears the DNA. If there is no methylation on both the strands of DNA, then RE cleaves
the DNA.

It is only by this methylation mechanism that, RE, although present in bacteria, does not cleave
the bacterial DNA but cleaves the foreign DNA. But there are some restriction enzymes which
function exactly in reverse mode. They cut the DNA if it is a methylate.

Examples

Examples of restriction enzymes include.

Enzyme Source Recognition Sequence Cut

5'GAATTC 5'---G AATTC---3'
EcoRI Escherichia coli
3'CTTAAG 3'---CTTAA G---5'
5'CCW GG 5'--- CCWGG---3'
EcoRII Escherichia coli
3'GGWCC 3'---GGW CC ---5'
5'GGATCC 5'---G GATCC---3'
BamHI Bacillus amyloliquefaciens
3'CCTAGG 3'---CCTAG G---5'
HindIII Haemophilus influenzae 5'AAGCTT 5'---A AGCTT---3'
 3'TTCGAA 3'---TTCGA A---5'
5'---T CGA---3'
5'TCGA
TaqI Thermus aquaticus 3'---AGC T---5'
3'AGCT

E. coli DNA Polymerase I

The E. coli DNA polymerase I is a DNA-dependent DNA polymerase that possesses both 3' -> 5' and 5'
-> 3' exonuclease activities.
It is a single-chain protein with a mass of about 109,000 Da that requires
magnesium as a cofactor. Each of its three enzymatic activities are encapsulated into distinct domains of
the holoenzyme, such that proteolytic deletions can be generated that lack one or more of the activities.
The so-calledKlenow fragmentis one such molecule that is widely used in recombinant DNA work.

DNA polymerase I was used frequently in the early days of recombinant DNA technology for
radiolabeling DNA and synthesizing cDNA. However, other enzymes have proven to be more effective
for these purposes, including a proteolytic fragment of DNA polymerase I called Klenow fragment and
T4 DNA polymerase
.
RNase P- It specifically cleaves at the 5’ end of RNA. It is a complex enzyme consisting of small
protein (20 kilodaltons) and a 377 -nucleotide RNA molecule. It has been observed that the RNA
molecule possesses at least part of the enzymatic activity of the complex. Hence, it is an example of
ribozyme.
Kinase- Kinase is the group of enzymes, which adds a free pyrophosphate (PO 4) to a wide variety
of substrates like proteins, DNA and RNA.

It uses ATP as cofactor and adds a phosphate by breaking the ATP into ADP and pyrophosphate. It is
widely used in molecular biology and genetic engineering to add radiolabelled phosphates
.

APPLICATIONS OF GENETIC ENGINEERING

The first genetically engineered drug was human
insulin, approved by theUnited StatesFood and Drug
Administrationin 1982. Another early application of genetic engineering was to create human growth
hormone as replacement for a drug that was previously extracted from human cadavers.
1986 the
In
FDA approved the first genetically engineered vaccine for humans,
hepatitis
for B. Since these early
uses of the technology in medicine, the use of GE has expanded to supply many drugs and vaccines.

One of the best known of genetic engineering is the creation

genetically
of modified organisms
(GMOs)
such as foods and vegetables that resist pest and bacteria infection and have longer freshness.

There are potentially momentous

biotechnologicalapplications of GM, for example oral
vaccines
produced naturally in fruit, at very low cost.

An ambition of some GE human

is enhancement
via genetics, eventually by
molecular engineering
. See
also: transhumanism

Medicines and Cures

The use of rDNA allows scientists to produce many products that were previously available only in
limited quantities: for example, insulin, which we referred to earlier. Until the 1980s the only source of
insulin for people with diabetes came from animals slaughtered for meat and other purposes. The supply
was never high enough to meet demand, and this drove up prices. Then, in 1982, the U.S. Food and
Drug Administration (FDA) approved the sale of insulin produced by genetically altered organisms—
the first such product to become available. Since 1982 several additional products, human
such as
growth hormone
, have been made with rDNA techniques

Cloning

A clone is a cell, group of cells, or organism that contains genetic information identical to that of the
parent cell or organism. It is a formasexual
of reproduction
(see Reproduction), and as such it is not as
new as it seems; what
is new, however, is humans' ability to manipulate cloning at the genetic level.
The first clones produced by humans as long as 2,000 years ago were plants developed
grafts
from
and
stem cuttings. By cloning—a process that calls into play complex laboratory techniques and the use of
DNA replication—people usually mean a relatively recent scientific advance. Among these techniques
is the ability to isolate and copy (that is, to clone) individual genes that direct an organism's
development.
 References

• TEXT BOOKS:-
 1.Biotechnology-expanding horizons: b.d.singh. Kalyani
publishers . nd
2 edition
nd
 2. Molecular biotechnology: s.b.parimrose. Panima publication. 2
edition

• INTERNET:-
 www. Google.com
 www.b ionewsonline.com

 www.library.thinkquest.org

 en.wikipedia.org/enzymes used in genetic engineering

 www.molecular-plant-biotechnology.info/plant
biotechnology
 www.yahoo.com
 www.biotech.about.com
/od/whatisbiotechnology
www.g eneticengineering.org
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