SUBJECT CODE:-CONCEPTS OF BIOTECHNOLOGY

TOPIC: - ENZYMES USED IN GENETIC ENGINEERING

SUBJECT - BTC012
SUBMITTEDTO: -

SUBMITTED BY:-

MR.HARSH

ROLL NO.R108B30

(LECT. IN BIOTECH)

REG.NO.1040070105
B.TECHBIOTECH
3RD SEM

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

What 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 klenow 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 5end 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 DNA technology, genetic modification/manipulation
(GM) and gene splicing are terms that apply to the direct manipulation of an organism's
genes. Genetic engineering is different from traditional breeding, where the organism's
genes are manipulated indirectly; genetic engineering uses the techniques of molecular
cloning and transformation to 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 of erythropoietin in
hamster ovary 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 as gene splicingis 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 a microorganism 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 an affinity for complementary ends 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 in fetal cells and to
diagnose certain blood disorders, such as sickle 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 to surmount 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 called lambda 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 ligase is a special type of ligase that can link together two DNA strands that
have single-strand breaks (a break in both complementary strands of DNA). The alternative, a doublestrand break, is fixed by a different type of DNA ligase using the complementary strand as a template
but still requires DNA ligase to create the final phosphodiester bond to fully repair the DNA.

DNA ligase has applications in both DNA repair and DNA replication. In addition, DNA ligase has
extensive use in molecular biology laboratories for Genetic 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 AMP + 2Pi) and then AMP binds to the enzyme to form the enzymeAMP

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 4C for better results.

Ligase mechanism
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends
of one nucleotide with the 5' phosphate end of 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 nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits
of nucleic acids. Older papers may use terms such as "polynucleotidase" or "nucleodepolymerase".
In 1960s, scientists Stewart Linn and Werner Arber isolated examples of the two types of enzymes
responsible for phage growth restriction in Escherichia coli (E. coli) bacteria. One of these enzymes
added a methyl group to 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 "methylase" and the other a "restriction nuclease"..

HindII enzyme 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 both DNA backbones

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 as the "recognition
sequence" for HindII.
HindII is only one example of the class of enzymes known as restriction nucleases. EcoRI comes from
Escherichia coli RY13 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 term "endonuclease"
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 an exonuclease.

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 two sugar-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
e

Source

EcoRI Escherichia coli

Recognition Sequence

Cut

5'GAATTC

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

3'CTTAAG

3'---CTTAA G---5'

When 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 endsboth 3' and 5'are sometimes called "sticky 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 I cleaves 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 radiolabeling by nick translation.

Ribonuclease A

is 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 phosphatase removes 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 been dephorphorylated or 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 restriction endonuclease) is an enzyme that cuts double-stranded or single
stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes,
found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against
invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a
process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it
from the restriction enzymes activity.
Different restriction enzymes that recognize the same sequence are known as neoschizomers. 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 as an isoschizomer.

Type I

Type I restriction enzymes were the first to be identified and are characteristic of two different strains
(K-12 and B) of E. 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 (ATP) and magnesium (Mg2+) ions, are required for their activity.
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 fragments sticky ends
of complementary DNA. 3. The hybrid plasmid is reincorporated into the bacterial cell, where it
replicates as part of the cells DNA. 4. A large number of daughter cells can be cultured and their gene
products extracted for human use.

Typical type II restriction 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 only Mg 2+ 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

or

-OH,

based

upon

the

terminal

molecule

(5

-PO4

or

-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

EcoRI Escherichia coli

EcoRII Escherichia coli
BamHI Bacillus amyloliquefaciens
HindIII Haemophilus influenzae

Recognition Sequence
5'GAATTC
5'---G

Cut
AATTC---3'

3'CTTAAG
5'CCWGG

3'---CTTAA G---5'
5'--- CCWGG---3'

3'GGWCC
5'GGATCC

3'---GGWCC ---5'
5'---G GATCC---3'

3'CCTAGG
5'AAGCTT

3'---CCTAG G---5'
5'---A AGCTT---3'

3'TTCGAA
TaqI

Thermus aquaticus

5'TCGA

3'---TTCGA A---5'
5'---T CGA---3'
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-called Klenow fragment is 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 the United States Food and Drug
Administration in 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. In 1986 the
FDA approved the first genetically engineered vaccine for humans, for hepatitis 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 of genetically modified organisms (GMOs)
such as foods and vegetables that resist pest and bacteria infection and have longer freshness.
There are potentially momentous biotechnological applications of GM, for example oral vaccines
produced naturally in fruit, at very low cost.
An ambition of some GE is human 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, such as human
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 form of asexual 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 from grafts and stem
cuttings. By cloninga process that calls into play complex laboratory techniques and the use of DNA

replicationpeople 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 . 2nd edition

2. Molecular biotechnology: s.b.parimrose. Panima publication. 2 nd
edition

INTERNET: www. Google.com

www.bionewsonline.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.geneticengineering.org