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1.

0 Introduction
Virtually all microbial traits are controlled or influenced by heredity. The inherited traits of microbes
include their shape and structural features, their metabolism. their ability to move or behave in various
ways, and their ability to interact with other organisms-perhaps causing disease. Individual organisms
transmit these characteristics to their offspring through genes

In 1866, the Czech-Austrian monk Gregor Mendel determined that traits are inherited as physical units,
now called genes. The precise function of genes, however, was not revealed until 1941, when George
Beadle and Edward Tatum published a scientific paper reporting that genes direct the production of
enzymes. Microorganisms, especially bacteria, have significant advantages as model organisms, in part
because of their unique characteristics.. The early work of Fred Griffith in 1928 on the transfer of
virulence (that is, the ability to cause disease) in the pathogen Streptococcus pneumoniae, commonly
called pneumococcus, set the stage for research showing that DNA was indeed the genetic material.
Oswald Avery and his colleagues then set out to discover which constituent in the heat-killed virulent
pneumococci was responsible for transformation.
Eight years later, Alfred Hershey and Martha Chase wanted to know if protein or DNA carried the
genetic information of a bacterial virus called T2 bacteriophage.

Microbial genetical terms

Codon A series of three nucleotides that code for a specific amino acid.

DNA Polymerase Enzyme that synthesizes DNA, using an existing strand as a


template to make a new complementary strand.

DNA Replication Duplication of a DNA molecule.


Chromosomes are structures containing DNA that physically carry hereditary
information; the chromosomes contain the genes

Gene The functional unit of the genome; it encodes a product, most often a
protein.

Genome Complete set of genetic information in a cell or a virus.

The genotype of an organism is its genetic makeup, the information that codes
for all the particular characteristics of the organism. The genotype represents
potelltial properties, but not the properties themselves.

Phenotype refers to actual, expressed properties, such as the organism's ability to


perform a particular chemical reaction. Phenotype, then, is the manifestation of
genotype.

Messenger RNA (mRNA) Type of RNA molecule translated during protein


synthesis.

Promoter Nucleotide sequence to which RNA polymerase binds to start


transcription.

Ribosomal RNA (rRNA) Type of RNA molecule present in ribosomes.

Ribosome Structure that facilitates the joining of amino acids during translation;
composed of ribosomal RNA (rRNA) and protein.

RNA Polymerase Enzyme that synthesizes RNA using one strand of DNA as a
template.

Transcription The process by which the information encoded in DNA is copied into
RNA.
Transfer RNA (tRNA) Type of RNA molecule involved in interpreting the genetic
code; each tRNA molecule carries a specific amino acid.

Translation The process by which the information carried by mRNA is used to


synthesize the encoded protein.
Characteristics of DNA

DNA is usually a double-stranded, helical structure. Each strand is


composed of a chain of deoxyribonucleotide subunits. Each nucleotide
contains a 5-carbon sugar (deoxyribose), a phosphate group, and one of
four different nucleobases (A, T, G, or C. The two strands of DNA are
complementary and are held together by hydrogen bonds between the
nucleobases. Wherever an adenine (A) is in one strand, a thymine (T) is
in the other; these opposing A-T bases are held together by two
hydrogen bonds. Similarly, wherever a guanine (G) is in one strand, a
cytosine (C) is in the other. These G-C bases are held together by three
hydrogen bonds, a slightly stronger attraction than that of an A-T pair.
The characteristic bonding of A to T and G to C is called base-pairing.
Because of the rules of base-pairing, one strand can always be used as a
template for the synthesis of the opposing strand. they are also
antiparallel.

Characteristics of RNA

R NA, like DNA, is composed of a chain of nucleotides.the sugar in the


nucleotides of RNA is ribos. RNA contains the nucleobase uracil in place
of the thymine found in DNA. Also, RNA is usually a single-stranded
linear molecule much shorter than DNA. In making the RNA molecule,
or transcript, the base-pairing rules apply except that uracil, rather
than thymine, pairs with adenine.

DNA replication
DNA replication is very important in gene expression

DNA molecule is a helical structure made up of 2 strands with


complementary bases joined together. Before DNA can be replicated it
requires a number of enzymes and protein.

Helicase
Single strand binding protein (SSB)
Primase
DNA polymerase
Exonuclease
DNA ligase
Helicase unwinds the DNA at a specific site called replication fork

Single strand binding protein stabilize the two separate strand in other to stop
them from annealing( joining back together)

Primase add a short segment of RNA called a primer so that DNA polymerase can
begin addition of nucleotide

Note; the directionality of replication is read up and write down from 3 to 5 prime

DNA polymerse adds bases complementary to the template strands

Single strand binding proteins (SSB) will be displace and move away. The new
strand will be produce in the 5 prime to 3 prime direction. And this process is
continues only in the leading strand

In the lagging strand DNA polymerase is unable to add up bases from 5 prime to 3
prime. To do that they need a new primer and a new DNA polymerase that will
add a short fragment of DNA called okazaki fragments. After adding a primer and
okazaki fragments an enzyme called exonuclease will eliminate the primer and
DNA polymerase will continue to add bases to the existing DNA. DNA polymerase
is incapable of joining with an already existing strand of newly synthesize DNA.

DNA ligase in an ATP dependent manner joins the two short DNA fragments or
components together, Note; the synthesis of two doughter DNA is semi
conservative.

Transcription
Transcription begins when RNA polymerase recognizes and binds to a promoter
region on the double-stranded DNA molecule.

Sigma factor binds to RNA polymerase and RNA polymerase binds to promoter to
form a closed promoter complex

Transcription begins when sigma factor dissociates from RNA polymerase by


forming an open complex. In the elongation phase, the RNA polymerase begins
the addition of ribonucleotides from 3 prime end of DNA template. The RNA
molecule is synthesized in the 5„ to 3„ direction as the RNA polymerase adds
nucleotides to the 3„OH group at the end of the growing chain.

Termination of transcription occurs when a protein called rho protein attaches


itself to the growing RNA chain

It drags itself to a region, RNA polymerase encounters a terminator, it falls off the
DNA template and releases the newly synthesized RNA.

Translation

Materials that are required in translation includes

1 mRNA which carries the codons


2 tRNAwhich carries the a.acid
3 Ribosome is involve in catalysis of the whole reaction
4 Translation factors (IF, EF and RF)
Translation initiation begins with the formation of 30s and 70s ribosomal complex
with the help of initiation factors (IF1,IF2,IF3).The ribosome contains three site (E,
P and A site). mRNA is attached in between the two complex

The first modified a. acid called F-met carried by tRNA will be attached to P-site.
In elongation, the second a. acid will be carried by the tRNA and attached it to the
A- site. When the second a. acid is in the A-site, ribosome will move along one
codon unit. The a. acid ( F-met) in p-site will form peptidyl bond with the amino
acid in A-site by an enzymes called peptidyl transferase

The tRNA that carries a. acid and an anticodon will bind with codon in mRNA.

Translocation of a. acid by the elongation factor EFGU will continue to occur from
A to P and P to E-site which will be exited.

The process of translocating the synthesis of the polypeptide chain will continue
until it reaches the stop codon UAA, UAA and UAG.

When they reaches the stop codon, all the structures ie (30s 50s), translation
factors and the tRNA will be dissociated for another synthesis to begin.
2.0 Bacterial Gene Regulation

In bacterial cells, many genes are routinely expressed, but others are
regulated in response to environmental conditions. A regulatory
mechanism sometimes controls the transcription of only a limited
number of genes, but in other cases, a wide array of genes is controlled
coordinately. A set of regulated genes transcribed as a single mRNA
molecule, along with the sequences that control its expression, is called
an operon. One of the most well-characterized examples is the lac
operon, which encodes proteins required for transporting and
hydrolyzing the disaccharide lactose.

■ Constitutive. Constitutive enzymes are synthesized constantly; the


genes that encode these enzymes are always active. For example, the
enzymes of glycolysis are constitutive.

■ Inducible. Inducible enzymes are not routinely produced at


significant levels; instead, their synthesis can be turned on when
needed. An example is b-galactosidase, the enzyme that hydrolyzes
lactose into its component monosaccharides—glucose and galactose

■ Repressible. Repressible enzymes are produced routinely, but their


synthesis can be turned off when they are not required. Repressible
enzymes are generally involved in biosynthetic (anabolic) pathways,
such as those that produce amino acids.

Mechanisms to Control Transcription

The methods a cell uses to prevent or facilitate transcription must be


readily reversible. Two of the most common regulatory mechanisms are
alternative sigma factors and DNA-binding proteins.

Alternative Sigma Factors As described earlier, sigma factor is a loose


component of RNA polymerase that functions in recognizing specific
promoters. Standard sigma factors recognize promoters for genes that
need to be expressed during routine growth conditions, but a cell can
also produce alternative sigma factors. These recognize different sets
of promoters, thereby controlling the expression of specific groups of
genes.

DNA-Binding Proteins Transcription is often controlled by proteins


that bind to specific DNA sequences. When a regulatory protein
attaches to DNA, it can act either as a repressor, which blocks
transcription, or an activator, which facilitates transcription.
Repressors A repressor is a regulatory protein that blocks
transcription (negative regulation). It does this by binding to an
operator, a specific DNA sequence located immediately downstream of
a promoter. When a repressor is bound to an operator, RNA
polymerase cannot progress past that DNA sequence. Repressors are
allosteric proteins, however, meaning that specific molecules can
attach to them and change their shape. This can change the repressor’s
ability to bind to operator DNA. there are two general mechanisms by
which different repressors can function:

Induction. The repressor is synthesized as a form that binds to the


operator, blocking transcription. When a molecule called an inducer
attaches to the repressor, the shape of the repressor changes so that it
can no longer attach to the operator. With the repressor unable to bind
to DNA, RNA polymerase may transcribe the gene.

Repression. The repressor is synthesized as a form that cannot bind to


the operator. However, when a molecule termed a corepressor
attaches to the repressor, the corepressor-repressor complex can then
bind to the operator, blocking transcription.

Activators

An activator is a regulatory protein that facilitates transcription


(positive regulation). Genes controlled by an activator have an
ineffective promoter preceded by an activator binding site. The binding
of the activator to the DNA enhances the ability of RNA polymerase to
initiate transcription at that promoter. Like repressors, activators can
be changed by the binding of other molecules. When a molecule called
an inducer binds to an activator, the shape of the activator is changed
so that it can now bind to the activator-binding site. Thus, the term
“inducer” applies to a molecule that turns on transcription, either by
stimulating the function of an activator or interfering with the function
of a repressor.

The lac Operon as a Model

Originally described in the early 1960s by François Jacob and Jacques


Monod, the lac operon of E. coli has served as an important model
for understanding the control of bacterial gene expression. This operon
encodes proteins involved with the transport and degradation of
lactose and is only turned on when glucose is not available but lactose
is. In this situation, the cell is forced to use lactose. When glucose is
available, it prevents expression of the lac operon genes, ensuring that
cells use the most efficiently metabolized carbon source (glucose) first.

Lactose and the lac Operon The lac operon uses a repressor that
prevents transcription when lactose is not available; the repressor
binds the operator, blocking RNA polymerase. When lactose is in the
cell, however, some of it is converted to allolactose, an inducer. This
compound binds the repressor and, in doing so, changes the repressor’s
shape so that it can no longer bind to the operator. With the operator
unoccupied, RNA polymerase can begin transcribing the operon.
However, this can happen only if glucose is not available in the growth
medium.

3.0 Recombinant DNA technology


Biotechnology uses microbiological and biochemical techniques to solve practical
problems and produce useful products. Today, recombinant DNA techniques
have made it possible to genetically alter organisms to give them more useful traits.
Researchers can isolate genes from one organism, manipulate the purified DNA in
vitro, and then transfer the genes into another organism. In fact, biotechnology is
now nearly synonymous with genetic engineering , the process of deliberately
altering an organism’s genetic information using in vitro techniques. S ince the
development of recombinant DNA techniques, a virtual toolbox of DNA
technologies has been created. The information and innovations these have
generated affect society in numerous ways—from agricultural practices and
medical diagnoses to evidence used in courtrooms.

Glosary of rDNA terms

Fluorescence in situ Hybridization (FISH) Technique used to detect a given


nucleotide sequence within intact cells on a microscope slide.

Genetic Engineering Deliberately altering an organism’s genetic information


using in vitro techniques.
Polymerase Chain Reaction (PCR) In vitro technique used to repeatedly
duplicate (amplify) a specific region of a DNA molecule, increasing the number of
copies exponentially.

Recombinant DNA Molecule DNA molecule created by joining DNA fragments


from two different sources.

Restriction Enzyme Type of enzyme that recognizes a specific nucleotide


sequence and then cuts the DNA within or near that site.

Vector DNA molecule, often a plasmid, that functions as a carrier of cloned


DNA.

Colony Blotting Technique used to determine which colonies on an agar plate


contain a given nucleotide sequence.

DNA Cloning Procedure in which a fragment of DNA is inserted into a vector


and then transferred into another cell, where it then replicates.

DNA Gel Electrophoresis A procedure used to separate DNA fragments


according to their size.

DNA Probe Single-stranded piece of DNA, tagged with an identifiable marker,


that is used to detect a complementary sequence.

DNA Sequencing Process of determining the nucleotide sequence of a DNA


molecule.

Steps in Recombinant DNA Technology


(i) Selection and isolation of DNA insert:
First step in rec DNA technology is the selection of a DNA segment of interest
which is to be cloned. This desired DNA segment is then isolated enzymatically.
This DNA segment of interest is termed as DNA insert or foreign DNA or target
DNA or cloned DNA.
(ii) Selection of suitable cloning vector:
A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to
be integrated. A suitable cloning vector is selected in the next step of rec DNA
technology. Most commonly used vectors are plasmids and bacteriophages.

(iii) Introduction of DNA-insert into vector to form recDNA molecule:

The target DNA or the DNA insert which has been extracted and cleaved
enzymatically by the selective restriction endonuclease enzymes [in step (i)] are
now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA
molecule which is often called as cloning-vector-insert DNA construct.

(iv) rec DNA molecule is introduced into a suitable host:

Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)]
is introduced into these host cells. This process of entry of rec DNA into the host
cell is called transformation. Usually selected hosts are bacterial cells like E. coli,
however yeast, fungi may also be utilized.

(v) Selection of transformed host cells:

Transformed cells (or recombinant cells) are those host cells which have taken up
the recDNA molecule. In this step the transformed cells are separated from the
non-transformed cells by using various methods making use of marker genes.

(vi) Expression and Multiplication of DNA insert in the host:


Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is
expressing the desired character in the host cells. Also, the transformed host cells
are multiplied to obtain sufficient number of copies. If needed, such genes may
also be transferred and expressed into another organism.

Tools for Recombinant DNA Technology


DNA technology utilizes a number of biological tools to achieve its objectives,
most important of them being the enzymes.

Important biological tools for rec DNA technology are:


Enzymes
Restriction Endonucleases
Exonucleases
DNA ligases
DNA polymerase
Cloning Vector
Host organism
DNA insert or foreign DNA
Linker and adaptor sequences
Enzymes
A number of specific enzymes are utilized to achieve the objectives of rec DNA
technology. The enzymology of genetic engineering includes the following types
of enzymes:
Restriction Endonuclease:
These enzymes serve as important tools to cut DNA molecules at specific sites,
which is the basic need for rec DNA technology

Exonuclease

Is an enzyme that removes nucleotides from the ends of a nucleic acid


molecule.An exonuclease removes nucleotide from the 5′ or 3′ end of a DNA
molecule. An exonuclease never produces internal cuts in DNA.

DNA ligase:

The function of these enzymes is to join two fragments of DNA by synthesizing the
phosphodiester bond. They function to repair the single stranded nicks in DNA
double helix and in rec DNA technology they are employed for sealing the nicks
between adjacent nucleotides.

DNA polymerases
These are the enzymes which synthesize a new complementary DNA strand of an
existing DNA or RNA template. A few important types of DNA polymerases are
used routinely in genetic engineering. One such enzyme is DNA polymerase

Cloning vector
It is another important natural tool which geneticists use in rec DNA technology.
The cloning vector is the DNA molecule capable of replication in a host organism,
into which the target DNA is introduced producing the rec DNA molecule.Different
types of DNA molecules may be used as cloning vehicles such as plasmids,
bacteriophages, cosmids.
Host organism
A good host organism is an essential tool tor genetic engineering. Most widely
used host for rec DNA technology is the bacterium E. coli. because cloning and
isolation of DNA inserts is very easy in this host. A good host organism is the one
which is easy to transform and in which the replication of rec DNA is easier. There
should not be any interfering element against the replication of rec DNA in the
host cells

DNA Insert Or Foreign DNA

The desired DNA segment which is to be cloned is called as DNA insert


or foreign DNA or target DNA. The selection of a suitable target DNA is
the very first step of rec DNA technology. The target DNA (gene) may be
of viral, plant, animal or bacterial origin. Following points must be
kept in mind while selecting the foreign DNA

It can be easily extracted from source.


It can be easily introduced into the vector.
The genes should be beneficial for commercial or
research point of view.
A number of foreign genes are being cloned for benefit of human
beings. Some of these DNA inserts are the genes responsible for the
production of insulin, interferon’s, lymphotoxins various growth factors,
interleukins, etc.

Linker and Adaptor Sequences

Linkers and adaptors are the DNA molecules which help in the
modifications of cut ends of DNA fragments. These can be joined to the
cut ends and hence produce modifications as desired.

Both are short, chemically synthesized, double stranded DNA


sequences. Linkers have (within them) one or more restriction
endonuclease sites and adaptors have one or both sticky ends. Different
types of linkers and adaptors are used for different purposes.

Linkers contain target sites for the action of one or more restriction
enzymes. They can be ligated to the blunt ends of foreign DNA or
vector DNA.

Adaptors are the chemically Synthesized molecules which have pre-


formed cohesive ends. Adaptors are employed for end modification in
cases where the recognition site for restriction endonuclease enzyme is
present within the foreign DNA.

Techniques Used In Recombinant DNA


Technology
A number of techniques are used for various purposes during different
steps of rec DNA technology. Such techniques serve for the fulfilment of
different requirements or to obtain proper information for drawing an
exact inference during genetic engineering. Some of these important
techniques are gel electrophoresis, blotting techniques, dot-blot
hybridization, DNA sequencing, artificial gene synthesis, polymerase
chain reaction, colony hybridization, etc.

Gel Electrophoresis
It is the technique of separation of charged molecules (in aqueous
phase) under the influence of an electrical field so that they move on
the gel towards the electrode of opposite charge i.e., cations move
towards the negative electrode and anions move towards the positive
electrode.
Blotting Techniques
Visualization of a specific DNA (or RNA or protein) fragment out of
many molecules requires a technique called blot transfer. In this
technique, the separated bands are transferred onto a nitrocellulose
membrane from the gel. Mainly there are three types of blot transfer
procedures:
Southern Blotting, Northern Blotting and Western blotting

DNA Sequencing
The segments of specific DNA molecules obtained by recombinant DNA
technology can be analysed for determining their nucleotide sequence.
The methods commonly used for DNA sequencing are:

 Enzymatic method or Sanger’s Dideoxy method.


 Chemical method or Maxam-Gilbert Method.
 Automated method.
Polymerase chain reaction(PCR)

A PCR-amplification cycle consists of three basic steps. They are


denaturation, annealing, and primer extension. In the denaturation
step, the template DNA is heated to 94°C for one minute or up to five
minutes. At this high temperature the DNA undergoes complete
denaturation and the double-stranded DNA (dsDNA) becomes single-
stranded.

The next step is the primer annealing. In this step the two primers, the
forward primers and the backward primers, anneal or hybridize to the
single-stranded template DNA at its complementary regions. Annealing
is usually carried out at a lower temperature depending on the length
and sequence of the primers. In standard cases it is in the range of 40
to 50°C.

The final step in each cycle is the extension in which the Taq DNA
polymerase synthesizes the DNA region between the primers using
dNTPs and Mg2+. The optimum temperature for carrying out the
primer extension reaction or polymerization of dNTPs is standardized at
72°C.
Main Requirements of PCR
Two nucleotide primers which are complementary to 3′ ends of

target DNA strands, Target DNA sequence, A heat stable DNA


polymerase e.g. Taq polymerase, Deoxy adenosine triphosphate
(dNTPs), A thermal cycler in which PCR is carried out

Main Types of PCR


1. Inverse PCR 2. Anchored PCR 3. Asymmetric PCR 4. Overlap-
Extension PCR

Uses of PCR:

1. For amplification of DNA


2. To detect mutations
3. To diagnose genetic disorders
4. To produce in vitro mutations
5. For preparing DNA for sequencing
6. To analyse genetic defects in single cells from human
embryos.
7. To identify virus & bacteria in infectious diseases.
8. For characterization of genotypes.

Colony Hybridization Technique


This technique is used in genetic engineering for the identification of
transformed bacterial cells (i.e. cells which contain foreign DNA). After
transformation of cells with a specific DNA, it is likely that only some of
those cells may have foreign DNA. For further procedure, firstly it is
important to screen such cells which are having foreign DNA.

This screening is done by using the technique of colony hybridization in


case of bacterial cells. A similar technique namely Plaque Hybridization
is utilized for screening of transformed bacteriophages.

Applications of Recombinant DNA


Technology
Genetic engineering or rec DNA technology has enormous and wide-
spread applications in all the fields of biological sciences. Genetic
engineering and biotechnology will continue to contribute in the future

to medicine, industry, and agriculture, as well as to basic research .


Medical Applications

The production of medically useful proteins such as somatostatin,


insulin, human growth hormone, and some interferons (signaling
molecules of the immune system) is of great practical importance.

 Production of Hormones:
By the advent of techniques of rec DNA technology, bacterial cells like
E.coli are utilized for the production of different fine chemicals like
insulin, somatostatin, somatotropin and p-endorphin. The genes of
interest are incorporated into the bacterial cells which are then cloned.
Such clones are capable of producing a fair amount of hormones like
insulin which have great commercial importance.

Biosynthesis of Interferon
By recDNA technolology method, the gene of human fibroblasts (which
produce interferon’s in human beings) is inserted into the bacterial
plasmid.

These genetically engineered bacteria are cloned and cultured so that


the gene is expressed and the interferon’s are produced in fairly high
quantities. This interferon, so produced, is then extracted and purified..

 Production of vaccines
A number of vaccines have been synthesized biologically through rec
DNA technology. DNA-vaccine is the preparation that contains a
gene encoding an immunogenic protein from the concerned
pathogen.

 Production of Antibiotics:
recDNA technology helps in increasing the production of antibiotics by
improving the microbial strains through modification of genetic
characteristics.

Application in Disease Prevention and


Diagnosis
Genetic engineering methods and techniques have greatly solved the
problem of conventional methods for diagnosis of diseases. It also
provides methods for the prevention of a number of diseases like AIDS,
cholera, etc. Monoclonal antibodies are useful tools for disease
diagnosis. Monoclonal antibodies are produced by using the technique

called hybridoma technology.

 Gene therapy
Gene therapy is undoubtedly the most beneficial area of genetic
engineering for human beings. It involves delivery of specific genes into
human body to correct the diseases. Thus it is the treatment of diseases
by transfer and expression of a gene into the patients’ cells so as to
ensure the restoration of a normal cellular activity.

On the basis of types of cells into which the functional genes are
introduced, the gene therapy may be classified as somatic gene therapy
and germ line gene therapy. Gene therapy is done either by using in
vivo strategy (also called as patient therapy) or by using the ex vivo

strategy.

Industrial application
 Application in Enzyme Engineering:
As we know that the enzymes are encoded by genes, so if there are
changes in a gene then definitely the enzyme structure also changes.
Enzyme engineering utilizes the same fact and can be explained as the
modification of an enzyme structure by inducing alterations in the
genes which encode for that particular enzyme.

 Production of Commercially Important Chemicals:


Various commercially important chemicals can be produced more
efficiently by utilizing the methods of rec DNA technology. A few of
them are the alcohols and alcoholic beverages obtained through
fermentation; organic acids like citric acid, acetic acid, etc. and vitamins
produced by microorganisms.

 Biofuel Production
Biofuels are derived from biomass and these are renewable and cost
effective. Genetic engineering plays an essentially important role in a
beneficial and large scale production of biofuels like biogas. bio
hydrogen biodiesel bio-ethanol., etc. Genetic engineering helps to
improve organisms for obtaining higher product yields and product

Application In agriculture
 Transgenic Plants:
A genetically modified plant, consisting in its genome, one or more
inserted genes of an unrelated plant is termed as a transgenic plant and
those inserted genes are called as transgenic. The development of
transgenic plant is possible by using recDNA technology, gene delivery
strategies and the tissue culture techniques.

Production of transgenic plants involves two main steps. transformation


of the target plant cells and then regeneration of transformed cells into
whole plants In the transformation step, foreign gene of interest is
introduced into the target plant cells.

This can be done by following any of the gene delivery systems available
like AMGT (Agrobacterium mediated gene transfer), using plant viruses
as vectors or by direct gene delivery system i.e., electroporation,
microinjection, particle-gun method, etc

 Production of Transgenic Animals


By the use of rec DNA technology, desired genes can be inserted into
the animal so as to produce the transgenic animal. commercially
important use of transgenic animals is the production of certain
proteins and pharmaceutical compounds. Transgenic animals also
contribute for studying the gene functions in different animal species.
Biotechnologists have successfully produced transgenic pigs, sheep, rats
and cattle.

Application in research
recDNA technology has an immense scope in Research and

Experimental studies. It is applied for:


 Localizing specific genes.
 Sequencing of DNA or genes.
 Study of mechanism of gene regulation.
 Molecular analysis of various diseases.
 Study of” mutations in DNA, etc.
Applications in forensic science:
The applications of rec DNA technology (or genetic engineering) in
forensic sciences largely depend on the technique called DNA profiling
or DNA fingerprinting. It enables us to identify any person by analysing
his hair roots Wood stains, serum, etc. DNA fingerprinting also helps to
solve the problems of parentage and to identify the criminals.

Application in environment
 Environment Protection
Genetic engineering makes its contributions to the environment
protection in various ways. Major approach in environment protection
is the use of recDNA technology for degradation of toxic pollutants
which harm the environment. Different microbes used for sewage
treatment, waste water treatment, industrial effluent treatment and for
bioremediation are greatly improved by genetic engineering practices
and thus present better results.

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