Recombinant DNA Technology (With Diagram)

In this article we will discuss about Recombinant DNA Technology:- 1.Steps in Recombinant DNA Technology 2. Tools for Recombinant DNA Technology 3. Techniques Used In Recombinant DNA Technology 4. Applications of Recombinant DNA Technology.

Steps in Recombinant DNA Technology:

Basic steps involved in rec DNA technology (or genetic engineering) are given below (Fig. 1):

i. Selection and isolation of DNA insert

ii. Selection of suitable cloning vector

iii. Introduction of DNA-insert into vector to form rec DNA molecule

iv. rec DNA molecule is introduced into a suitable host.

v. Selection of transformed host cells.

vi. Expression and multiplication of DNA-insert in the host.

(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:

1WDNA 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:

(A) Enzymes:

a. Restriction Endonucleases

b. Exonucleases

c. DNA ligases

d. DNA polymerase

(B) Cloning Vector

(C) Host organism

(D) DNA insert or foreign DNA

(E) Linker and adaptor sequences.

An account of all these biological tools of genetic engineering is given below:

(A) 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:

(a) Restriction Endonuclease:

These enzymes serve as important tools to cut DNA molecules at specific sites, which is the basic need for rec DNA technology.

These are the enzymes that produce internal cuts (cleavage) in the strands of DNA, only within or near some specific sites called recognition sites/recognition sequences/ restriction sites 01 target sites. Such recognition sequences are specific for each restriction enzyme. Restriction endonuclease enzymes are the first necessity for rec DNA technology.

The presence of restriction enzymes was first of all reported by W. Arber in the year 1962. He found that when the DNA of a phage was introduced into a host bacterium, it was fragmented into small pieces. This led him to postulate the presence of restriction enzymes. The first true restriction endonuclease was isolated in 1970s from the bacterium E. coli by Meselson and Yuan.

Another important breakthrough was the discovery of restriction enzyme Hind-II in 1970s by Kelly, Smith and Nathans. They isolated it from -the bacterium Haemophilus influenza. In the year 1978, the Nobel Prize for Physiology and Medicine was given to Smith, Arber and Nathans for the discovery of endonucleases.

Types of Restriction Endonucleases:

There are 3 main categories of restriction endonuclease enzymes:

Type-I Restriction Endonucleases

Type-II Restriction Endonucleases

Type-III Restriction Endonucleases

Type-I Restriction Endonucleases:

These are the complex type of endonucleases which cleave only one strand of DNA. These enzymes have the recognition sequences of about 15 bp length (Table 1).

They require Mg++ ions and ATP for their functioning. Such types of restriction endonucleases cleave the DNA about 1000 bp away from the 5′ end of the sequence ‘TCA’ located within the recognition site. Important examples of Type-I restriction endonuclease enzyme are EcoK, EcoB, etc.

Type-II Restriction Endonucleases:

These are most important endonucleases for gene cloning and hence for rec DNA technology. These enzymes are most stable. They show cleavage only at specific sites and therefore they produce the DNA fragments of a defined length. These enzymes show cleavage in both the strands of DNA, immediately outs.de then- recognition sequences. They require Mg⁺⁺ ions for their functioning.

Such enzymes are advantageous because they don’t require ATP for cleavage and they cause cleavage in both strands of DNA. Only Type II Restriction Endonucleases are used tor gene cloning due to their suitability.

The recognition sequences for Type-II Restriction Endonuclease enzymes are in the form of palindromic sequences with rotational symmetry, i.e., the base sequence .n the first half of one strand of DNA is the mirror image of the second half of other strand of that DNA double helix (Fig. 2). Important examples of Type-II Restriction endonucleases include Hinfl, EcoRI, PvuII, Alul, Haelll etc.

Type-III Restriction Endonucleases:

These are not used for gene cloning. They are the intermediate enzymes between Type-I and Type-II restriction endonuclease. They require Mg⁺⁺ ions and ATP for cleavage and they cleave the DNA at well-defined sites in the immediate vicinity of recognition sequences, e.g. Hinf III, etc.

Nature of cleavage by Restriction Endonucleases:

The nature of cleavage produced by a restriction endonuclease is of considerable importance.

They cut the DNA molecule in two ways:

i. Many restriction endonucleases cleave both strands of DNA simply at the same point within the recognition sequence. As a result of this type of cleavage, the DNA fragments with blunt ends are generated. PvuII, Haelll, Alul are the examples of restriction endonucleases producing blunt ends. Blunt ends may also be referred to as flush ends.

ii. In the other style of cleavage by the restriction endonucleases, the two strands of DNA are cut at two different points. Such cuts are termed as staggered cuts and this results into the generation of protruding ends i.e., one strand of the double helix extends a few bases beyond the other strand. Such ends are, called cohesive or sticky ends.

Such ends have the property to pair readily with each other when pairing conditions are provided. Another feature of the restriction endonucleases producing such sticky ends is that two or more of such enzymes with different recognition sequences may generate the same sticky ends.

(b) Exonucleases:

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.

In rec DNA technology, various types of exonucleases are employed like Exonuclease Bal31, E. coli exonuclease III, Lambda exonuclease, etc.

Exonculease Bal31 are employed for making the DNA fragment with blunt ends shorter from both its ends.

E coli Exonuclease III is utilized for 3’end modifications because it has the capability to remove nucleotides from 3′-OH end of DNA.

Lambda exonuclease is used to modify 5′ ends of DNA as it removes the nucleotides from 5′ terminus of a linear DNA molecule.

(c) 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. This enzyme is also termed as molecular glue.

(d) 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 ! which , prepared from E coli. The Klenow fragment of DNA polymerase-I .s employed to make the protruding ends double-stranded by extension of the shorter strand.

Another type of DNA polymerase used in genetic engineering is Taq DNA polymerase which is used in PCR (Polymerase Chain Reaction).

Reverse transcriptase is also an important type of DNA polymerase enzyme for genetic engineering. It uses RNA as a template for synthesizing a new DNA strand called as cDNA a e complementary DNA). Its main use is in the formation of cDNA libraries. Apart from all these above mentioned enzymes, a few other enzymes also mark their importance in genetic engineering.

A brief description of these is given below:

(a) Terminal deoxynucleotidyl transferase enzyme:

It adds single stranded sequences to 3′-terminus of the DNA molecule. One or more deoxynbonucleotides (dATP, dGTP, dl IP, dCTP) are added onto the 3′-end of the blunt-ended fragments.

(b) Alkaline Phosphatase Enzyme:

It functions to remove the phosphate group from the 5′-end of a DNA molecule.

(c) Polynucleotide Kinase Enzyme:

It has an effect reverse to that of Alkaline Phosphatase, i.e. it functions to add phosphate group to the 5′-terminus of a DNA molecule

(B) Cloning Vectors:

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.

A cloning vector may also be termed as a cloning vehicle or earner DNA or simply as a vector or a vehicle a great variety of cloning vectors are present for use with E. coli is the host organism.

However under certain circumstances it becomes desirable to use different host for cloning experiments. So, various cloning vectors have been developed based on other bacteria like Bacillus, Pseudomonas, Agrobacterium, etc. and on different eukaryotic organisms like yeast and other fungi.

The cloning vector which has only a single site for cutting by a particular restriction endormclease is Considered as a good cloning vector. Different types of DNA molecules may be used as cloning vehicles such as they may be plasmids, bacteriophages, cosmids, phasmids or artificial chromosomes.

(C) 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 winch 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

(D) 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:

1. CD It can be easily extracted from source.

2. It can be easily introduced into the vector.

3. 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.

(E) 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 (Fig. 5a). Then they undergo a treatment with a specific restriction endonuclease to produce cohesive ends of DNA fragments EcoRI-linker is a common example of frequently used linkers.

Adaptors are the chemically Synthesized molecules which have pre-formed cohesive ends (Fig. 5b). Adaptors are employed for end modification in cases where the recognition site for restriction endonuclease enzyme is present within the foreign DNA.

The foreign DNA is ligated with adaptor on both ends. This new molecule, so formed, is then phosphorylated at the 5′-terminii. Finally foreign DNA modified with adaptors is integrated into the vector DNA to form the recombinant DNA molecule.

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.

The genomic DNA is extracted from the desired host and is then fragmented using restriction endonucleases.

For separation of these cut fragments and isolation of desired DNA fragment, the technique of gel electrophoresis is employed. Gel electrophoresis may be of horizontal or vertical type. Usually agarose gel is used for separation of large segments of DNA while the polyacrylamide gel is used for the separation of small DNA fragments which are only a few base pairs long.

Gel electrophoresis employs a buffer system, a medium which is a gel and a source of direct current (Fig. 6). Samples having DNA fragments are applied on the gel and current is passed through the system for an appropriate time. Different DNA fragments move up to different distances on the gel depending on their charge to mass ratio.

The heavier fragments move a little, while the lighter DNA fragments move up to a larger distance. Following the migration of the molecules, the gel is treated with selective stains to show the location of separated molecules in the form of bands.

Very large DNA molecules or chromosomes cannot be separated even by Agarose Gel electrophoresis. For separation of such very large DNA molecules (sometimes representing whole chromosomes), a new technique is used which is known as Pulse Field Gel Electrophoresis (PFGE).

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.

Southern blotting is named after the person who devised this technique, viz. E.M. Southern (1975). The other names began as laboratory jargon but they are now accepted terms.

Technically, blotting may be defined as the transfer of macromolecules from the gel onto the surface of an immobilizing membrane like nitrocellulose membrane. It is to note here that during such transfer, the relative positions of bands (of macromolecules) are same on the membrane as they occurred on the gel.

The membranes which may.be used in blotting are nitrocellulose membrane, nylon membrane, carboxymethyl membrane, diazobenzyl-oxymethyl (DBM) membranes, etc.

Southern blotting is used for the transfer of DNA from gel onto the membrane while Northern and Western blotting are used for the transfer of RNA and protein bands respectively. One other blotting technique is south-western blotting which examines the protein-DNA interactions.

A schematic representation of southern blotting technique is given in the fig. 7. In this technique first of all, the sample DNA is digested with restriction enzymes to obtain fragments of different lengths. These differently sized DNA segments are then passed through Agarose Gel Electrophoresis for their separation based on their lengths.

The gel so obtained with different bands of DNA fragments is placed on top of buffer saturated filter papers which act as a filter paper wick. Above gel is put a nitrocellulose filter and over nitrocellulose filter are placed many dry filter paper sheets. With the movement of buffer towards the dry filter papers, the DNA bands are also moved upwards and hence they get bound to the nitrocellulose filter membrane.

Now, the nitrocellulose filter is removed and baked in vacuum. DNA fragments on the nitrocellulose filter are hybridized with single stranded radioactively labeled probes. Washing is done to remove unbound probes and finally the DNA bands with radioactivity are visualized by autoradiography.

In Northern Blotting, RNA molecules are blot transferred from the gel onto a chemically reactive paper. Western blotting is used for proteins and its working is based on the specificity of antibody-antigen reaction. In this technique the hybridization of bound proteins is done with radioactively labeled antibodies.

Dot Blot Hybridization:

The procedure of this technique is almost the same as blotting, but the only difference is that the DNA fragments are not separated by electrophoresis, instead they are directly applied as a dot on the nitrocellulose membrane.

Then radioactively labeled DNA probes having the complementary base sequences to the DNA of interest are applied on this membrane to allow its hybridization. The position of this hybridization is then detected by autoradiography method.

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:

i. Enzymatic method or Sanger’s Dideoxy method.

ii. Chemical method or Maxam-Gilbert Method.

iii. Automated method.

(i) Enzymatic method of DNA sequencing is also called as Sanger-Coulson method of sequencing of DNA molecules. This method involves the use of single stranded DNA as a template for DNA synthesis.

The dideoxynucleotide triphosphates (ddNTPs like ddCTP, ddGTP, ddATP, ddTTP) are incorporated in the growing chain and they terminate the chain synthesis because they are unable to form a phosphodiester bond with next deoxy-nucleotide triphosphate.

For sequencing, the reaction mixture is taken in four separate test tubes. In each test tube is added one particular ddNTP. As a result, different sizes of newly synthesized DNA strands are obtained in each test tube which are terminated by a particular ddNTP. These segments are then separated by electrophoresis and then the DNA sequences are obtained by reading the bands on autoradiogram from bottom to the top of gel.

(ii) Chemical method of DNA sequencing involves the degradation of DNA by using chemicals, rather than synthesis of new DNA. In this type of sequencing, the DNA sample is labeled radioactively at 3′ ends and separated into single strands. Sample is then divided into four test tubes, each treated with a specific chemical reagent which degrades only at specific nucleotide base like G or C or ‘A and G’ or ‘C and T’.

As a result of this partial chemical cleavage, a number of differently sized fragments are obtained in each test tube. These fragments are separated by gel electrophoresis and then observed under autoradiography to interpret the nucleotide sequence of sample DNA. Chemical method is not used very Commonly because it is a slow and labour intensive process.

(iii) Automatic DNA sequencing methods have been developed by improvements in dideoxy-method. A number of automatic DNA sequencing machines have also been invented which are capable of sequencing thousands of nucleotides within few hours.

Such methods involve the tagging of fluorescent dyes to ddNTPs, slab gel sequencing systems, capillary gel sequencing systems and PCR-based DNA sequencing techniques. Such techniques are faster and more reliable.

Artificial Gene Synthesis:

This technique may also be called as oligonucleotide synthesis. It is one of those techniques which have been adopted for the synthesis of desired gene or DNA fragment. Gene synthesis is now a routine laboratory procedure to be utilized in the rec DNA technology.

First success in the approach of artificial gene synthesis was achieved by Dr. Har Govind Khorana and his co-workers in 1970 when they synthesized the artificial gene for a t-RNA in vitro which had potential for functioning within a living cell.

Major approaches available for the artificial synthesis of genes are:

Enzymatic synthesis of Gene:

When details of base sequence of concerned gene are available, the polynucleotide of that same base sequence can be synthesized by enzymatic method. In this method the bacterial enzyme Polynucleotide phosphorylase is utilized. This method is easy to perform and does not require any template.

Chemical synthesis of Gene:

Once the base sequence of a gene is deducted, this gene can be synthesized by a purely chemical method as used by Khorana and his co-workers for the synthesis of gene for yeast alanyl t-RNA. This method utilizes different chemical reagents for various steps of the process.

There are mainly three distinct methods, which are phosphodiester phosphotriester, and phosphite-triester methods. These methods differ in their strategies for protecting the hydroxyl group of the phosphate residues.

If the detailed sequence of the concerned gene is unknown then the artificial gene is synthesized in the form of cDNA i.e. complementary DNA from the mRNA of that gene. In this method, the enzyme employed is RNA directed DNA polymerase.

PCR (Polymerase Chain Reaction):

PCR is a technique for the amplification (or cloning) of a target sequence of DNA. It is sometimes also referred to as in vitro gene cloning (without expression of that gene). PCR is an important technique in molecular biology and it was discovered by Kary Mullis in 1985 (Fig. 8). It is carried out in vitro and by it, upto billion copies of the target DNA sequence can be obtained from a single copy within few hours only.

Outline of PCR:

PCR is a technique which results in selective amplification of a selected DNA molecule. One limitation of PCR is that the border region sequences of the DNA (to be amplified) must be known in order to select the appropriate primers which anneal (attach) at its 3′ ends. Primer annealing is important due to the fact that enzyme DNA polymerases require double stranded (ds) primer regions for initiating the DNA synthesis.

The whole reaction of PCR takes place in a tube called eppendorf tube. Scientists are using PCR in a number of disciplines due to the advantages like it is a quick, simple and extremely accurate technique. Major limitation of PCR is that due to its extreme sensitivity it may produce erroneous results caused by several inhibitors or contaminating DNA segments present in the sample DNA preparation.

Main Requirements of PCR:

a. Two nucleotide primers which are complementary to 3′ ends of target DNA strands

b. Target DNA sequence.

c. A heat stable DNA polymerase e.g. Taq polymerase.

d. Deoxy adenosine triphosphate (dATP)

e. Deoxy thymidine triphosphate (dTTP)

f. Deoxy cytidine triphosphate (dCTP)

g. Deoxy guanosine triphosphate (dGTP)

h. A thermal cycler in which PCR is carried out

Steps in PCR:

A generalized PCR-protocol involves following steps (however, the temperature-time profile may vary according to the requirements):

(a) Mix target DNA sequence, excess of primers, dATP, dTTP, dCTP, dGTP and Taq polymerase in the reaction mixture in eppendorf tube. Place this tube in thermal cycler.

(b) Reaction mixture is given high temperature of about 90-98°C for few seconds to denature the DNA. As a result the double stranded DNA becomes single stranded.

(c) Temperature is changed to about 55°C for 20 seconds so that primers are annealed at 3′ ends of DNA.

(d) Now the temperature is maintained at 72°C for 30 seconds which facilitates the functioning of Taq polymerase thus synthesizing the complementary strand of DNA.

(e) Hence, one cycle of PCR is completed here resulting in the formation of two ds DNA molecules from one ds DNA.

(f) Same cycle is repeated till the required number of DNA copies are obtained.

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 (Fig. 9). A similar technique namely Plaque Hybridization is utilized for screening of transformed bacteriophages.

Basic principle of this technique lies in the in-situ hybridization of transformed bacterial cells with a radioactive probe sequence. Due to the specificity of probe, it enables rapid identification of one colony (through radioactivity) even amongst many thousands of colonies.

The transformed bacterial cells are first of all plated on a suitable agar plate which is termed as the master plate. Colonies are grown in the master plate. These colonies on the master plate are replica-plated onto a nitrocellulose or nylon membrane by placing it gently over the master plate. This replica-plate carrying the colonies is removed and treated with alkaline reagent to lyse the bacteria.

DNA of those bacterial cells is denatured. Proteins on the membrane are digested. Finally the membrane is washed to remove all other molecules, leaving behind only the denatured DNA bound to it, in the form of DNA print of the colonies.

This DNA print is then hybridized with a radioactively labeled RNA/DNA probe. Membrane is washed to remove any unbound probe and then autoradiography is done to detect radioactivity. The positions of the DNA prints showing up in autoradiograph are then compared with the master plate to identify the transformed colony.

Applications of Recombinant DNA Technology:

Genetic engineering or rec DNA technology has enormous and wide-spread applications in all the fields of biological sciences.

Some important applications of rec DNA technology are enlisted below:

(1) Production of Transgenic Plants:

By utilizing the tools and techniques of genetic engineering it is possible to produce transgenic plants or the genetically modified plants. Many transgenic plants have been developed with better qualities like resistance to herbicides, insects or viruses or with expression of male sterility, etc.

Also they allow the production of commercially important biochemical, pharmaceutical compounds, etc. Genetic engineering is capable of introducing the improved post-harvest characteristics in plants also. Transgenic plants also aid in the study of the functions of genes in plant species.

(2) 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. The method of rec DNA technology aids the animal breeders to increase the speed and range of selective breeding in case of animals. It helps for the production of better farm animals so as to ensure more commercial benefits.

Another 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.

(3) 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. Human Insulin Hormone i.e., Humulin is the first therapeutic product which was produced by the application of rec DNA technology.

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.

(4) Production of Vaccines:

Vaccines are the chemical preparations containing a pathogen in attenuated (or weakened) or inactive state that may be given to human beings or animals to confer immunity to infection. A number of vaccines have been synthesized biologically through rec DNA technology.

These vaccines are effective against numerous serious diseases caused by bacteria, viruses or protozoa. These include vaccines for polio, malaria, cholera, hepatitis, rabies, smallpox, etc. The generation of DNA vaccines has revolutionized the approach of treatment of infectious diseases. DNA-vaccine is the preparation that contains a gene encoding an immunogenic protein from the concerned pathogen.

(5) Biosynthesis of Interferon:

Interferon’s are the glycoproteins which are produced in very minute amounts by the virus-infected cells. Interferon’s have antiviral and even anti-cancerous properties. By recDNA technology 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.

(6) Production of Antibiotics:

Antibiotics produced by microorganisms are very effective against different viral, bacterial or protozoan diseases. Some important antibiotics are tetracyclin, penicillin, streptomycin, novobiocin, bacitracin, etc.

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

(7) 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.

(8) 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.

(9) Prevention and Diagnosis of Diseases:

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.

The monoclonal antibodies bear specificity against a specific antigen. These are used in the diagnosis of diseases due to their specificity. Genetic engineering allows the production of hybridoma which is a cell obtained by the fusion of a lymphocyte cell capable of producing antibodies and a single myeloma cell (tumour cell).

(10) 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.

(11) Practical Applications of Genetic Engineering:

recDNA technology has an immense scope in Research and Experimental studies.

It is applied for:

a. Localizing specific genes.

b. Sequencing of DNA or genes.

c. Study of mechanism of gene regulation.

d. Molecular analysis of various diseases.

e. Study of” mutations in DNA, etc.

(12) 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.

(13) 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 tolerance.

Genetically stable high producing microorganisms are being developed by using modern recDNA techniques, which aid in an efficient production of bioenergy.

The energy crop plants are those plants which use solar energy in a better way for production of biomass. Genetic improvements of these energy crop plants greatly help for quick and high Product on of biomass which in turn reduces the biofuel production cost. The fermenting microbes which are utilized for biogas production are improved at the genetic level for achieving better result.

(14) Environment Protection:

Genetic engineering makes its contributions to the environment protection in various ways. Most important to mention are the new approaches utilized for waste treatments and bioremediation Environment protection means the conservation of resources and hence to limit the degradation of environment.

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.

The plant species can also be developed by using various gene transfer techniques for acquiring better options for phytoremediation. Biological deodorization is a newer technology that involves the decomposition of bad stalling ingredients by microorganisms Genetic -peering play an equally sincere attention towards the improvisation and betterment of such deodorizing microbes.

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, that are: transformation of the target plant cells and then regeneration of transformed cells into whole plans 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. At present, the particle gun method and AMGT are the most favourable methods of gene delivery into plant cells.

Use of Marker Genes:

After the transformation step is over, it becomes essential to identify the transformed cells. Here, the role of marker genes is marked. The detection of integration of foreign genes into the plant genome requires the use of marker gene that either allow the transformed cells to be selected (selectable marker gene) or that encodes an activity which can be assayed or scored (scorable marker gene).

The use of a dominant selectable marker gene serves as a direct means of obtaining only transformed cells in culture because the non-transformed cells die on the selective medium. Therefore selectable marker genes are the frequently used marker genes. Usually, these selectable marker genes are introduced along with the foreign gene into the plant cells.

Although these marker genes are of immense utility in differentiating transformed cells from the non-transformed cells, but they pose some problems too. There is a threat of accidental transfer of resistance genes (used as selectable markers) to the pathogenic soil-bacteria which may cause disaster.

Secondly, the presence of a selectable marker gene makes it difficult to insert additional foreign genes into the transgenic plant as the same selectable markers gene cannot be used twice. To avoid these problems, a number of advanced strategies have been developed for the removal of marker genes and for the production of marker-free transgenic plants.

Production of transgenic crops has become a crucial part of plant breeding and it has immensely uplifted the possibilities of crop improvement programmes all over the world Numerous transgenic plants of different species have already been in cultivation and some are undergoing field trials.

They are beneficial because they contain improved agronomic traits which are commercially efficient. Transgenic crop plants have many beneficial traits like resistance against different pathogens pests, abiotic stresses, and improved nutritional quality higher yield, better phenotypes, etc. The first transgenic plant was generated in early 1980s when diverse foreign genes were introduced into tobacco plants.

Flavr-Savr tomato marketed in USA in 1994 by Calgene Co. was the first transgenic variety to reach the market. These transgenic tomatoes retain their freshness for long periods.

Freedom II squash marketed by Agrow Seed Co. resist the infection by viral diseases. High lauric rapeseed is an approved genetically engineered plant which produced rapeseeds rich in laurate (fatty-acid) which is useful in making soaps, detergents and shampoos.

Roundup Ready soya been developed by. Monosanto Co. is intended for making animal feed and not tor human use. Food and Drug Administration (FDA) of U.S.A. has so far approved a number of transgenic crops of plants species like rapeseeds, cotton, tomato, maize, sugar beet. papaya, soybean, etc., which have transgenes inserted for producing improved traits.

Some improved traits generally present in transgenic plants are one or more from the following (Table 2):

(i) Resistance to biotic stresses like:

a. Insect resistance

b. Resistance to weeds

c. Viral Resistance

d. Fungal Disease Resistance

e. Bacterial Disease Resistance

(ii) Herbicide Resistance:

(iii) Resistance against abiotic stresses like:

i. Drought

ii. Salinity

iii. Heat

iv. Frost

v. Metal toxicity

(iv) Modified quality of starch, edible-oils, proteins obtained from crop plants.

(v) Improved Nitrogen fixing capacity

(vi) Delayed ripening for improved storage and longer shelf-life.

(vii) Seedless fruits for better commercial values.

(viii) Improved colour, fragrance and longer life of commercial flowers.

Beyond possessing one or more of the above traits, transgenic plants are also utilized as bioreactors for manufacture of pharmaceutical chemicals and other commercial chemicals. These are also applied for studying the regulation of gene expressions under different conditions of factors like light and temperature (Table 2).

Transgenic research is being carried over in India also with a view to strengthen the plant biotechnology in country. Department of Biotechnology (DBT) makes funds for the promotion of transgenic research in India.

A number of institutes like Central Potato Research Institute (CPRI), Shimla; Indian Agricultural Research Institute (IARI), New Delhi; Central Rice Research Institute (CRRI), Cuttack; Delhi University, Punjab University, Ludhiana, etc. have made significant development in transgenic research producing a number of transgenic crop plants mainly in the species like rice, cotton, rapeseed and tobacco.

Molecular Farming:

This term describes the use of genetically modified plants for the production of scientifically, medically and/or industrially important biomolecules.

The concept behind molecular farming involves the growing and harvesting of plants with novel traits (i.e., transgenic plants) for producing biomolecules rather than food, feed and fibre. Potential biomolecules which can be produced through molecular farming include the medical products like pharmaceuticals (drugs), vaccines, diagnostic products, industrial chemicals, biodegradable plastics, biologies, etc.

Molecular farming has emerged as a promising industry having its base in plant biotechnology. It has attained a great importance in the field of pharmaceutical and industrial production because it ensures the cost-effective production of safe and functional products, which are expensive and difficult to be produced by any other means.

Molecular farming is actually an application of genetic engineering. It uses the genetically modified plants as ‘biological factories’ to produce recombinant protein products for a variety of uses.

In plant molecular farming, first of all the plants to be used as the ‘expression system’ are chosen. The organism into which the foreign gene is inserted for expression of the desired new product in molecular farming is called as expression system.

A foreign gene associated with the production of desired biomolecule is then integrated into plants’ genome. Such genetically engineered i.e., transgenic plants are then grown on agricultural scale providing them with water, sunlight and essential nutrients. During their growth, these transgenic plants synthesize the useful biomolecules which get accumulated in the plant tissues.

These plants are then harvested and the desired product is extracted and purified from the plants.

Some important examples of products that have been developed experimentally through plant molecular farming are interleukin in tobacco, edible vaccines, various enzymes for use in food processing, enzymes for treatment of human diseases, bio plastics from biodegradable molecules in corn, functional antibodies, hormones, blood proteins, trypsin and gastric lipase in corn, etc.

Molecular farming also involves the use of transgenic bacteria, plant viruses, yeasts, animal cell culture and transgenic animals as the expression systems for production of the desirable novel compounds.

But, as the plants offer numerous advantages over animals and animal cultures therefore, the molecular farming involving plants (i.e., plant molecular farming) is the most talked about and practically useful technique. A more closely related term is ‘pharming’ which is mostly applied for the use of ‘transgenic animals’ for the production of pharmaceuticals.

DNA Fingerprinting:

DNA fingerprinting is also called as DNA profiling. This technique was discovered in the year 1986 by British geneticist Alec Jeffrey’s of Leicester University. DNA fingerprinting aids in identification of individuals at the genetic level. It is a well-known fact that every living organism can be differentiated from the other only due to the sequence of nucleotides in the chromosome.

DNA profiling technology characterizes the segments of DNA which helps in the identification of individuals. Its basic requirement is the availability of samples like blood stains, semen, urine, tears, saliva, sweat, hair roots, etc.

For DNA fingerprinting, first of all, the DNA isolated from sample are digested with the help of suitable restriction enzymes.

This digested DNA is then subjected to electrophoresis and southern hybridization which involves its hybridization with a specific probe representing the highly variable region of the organism’s genome. As a result of this, polymorphism is generated. Due to this polymorphism it is very rare that two persons may have same pattern in DNA fingerprints.

This technique of DNA fingerprinting has revolutionized the field of forensic medicine as it is very beneficial for identification of criminals like murderers or rapists. This also helps in solving parentage disputes by identifying the real biological father of the child by analysing the DNA fingerprints of child and suspected father.

In cases where the samples of blood stain, semen etc. has partially degraded DNA the technique of PCR can be applied for amplification of DNA from sample. This enables better characterization of DNA which would not be possible otherwise. In India, facilities for DNA fingerprinting are available at CDFD (Centre for DNA Fingerprinting and Diagnostics), Hyderabad.

Mini-satellites which are tandem repeats of short sequences are used as probes while preparing DNA fingerprints of humans. In case of plants, such probes are not present, therefore, usually RFLPs (Restriction Fragment Length Polymorphism) or simple sequence repeats like (CT) or (AC)n etc. are used as probes for DNA fingerprinting for varietal identification.

GEMs:

GEM stands for Genetically Engineered Microbes. Those microorganisms which are modified through the use of genetic engineering techniques to fulfil specific needs are called as GEMs They are utilized for performing functions which would not be possible through the use of their natural counterparts.

For modifying microbes, foreign genes are introduced in o to genome using the recDNA technology, so as to obtain desired functioning of those microbes GEMs are of great use in different fields specially in industries Amongst different microbe.

The genetically engineered bacteria have found enormous utility in every field. Transfer and expression of beneficial genes into microorganisms has opened a new era for exploiting the microbial bioprocesses for attainment of better commercial outputs.

Some applications of GEMs in different areas are discussed below:

(i) GEMs specially bacterial strains are helpful for crop protection from diseases or abiotic stresses. Development of a virulent strains and antibiotic-producing strains of microbes are commonly used methods for crop protection.

(ii) Genetically engineered bacteria have also been applied for better crop production by enhancing their nitrogen fixation capacity. This is done by transferring of efficient nif genes into the bacterial genome. Rhizobium melilottii is a successful example in this case.

(iii) GEMs are better sources used for enzyme productions on commercial scales.

(iv) GEMs can also be applied for achieving enhanced production and quality of SCP and other compounds used as food and feed.

(v) A number of commercially important chemicals like amino acids, organic acids, ethanol antibiotics, etc. can be efficiently produced by utilizing GEMs. For example, genetically engineered strains of bacteria Bacillus amyloliquefaciens have been in use for large scale production of amino acids.

(vi) GEMs are very useful for abatement of environmental pollution. They have an immense potential for bioremediation of contaminants. Most prominent example for this « superbug which is an oil-eating bacteria Pseudomonas putida (patented in 1980) developed by an India born American scientist Dr. Ananda Mohan Chakraborty.

Splicing Genes:

It is an important step in gene cloning/genetic engineering. It is actually a gene manipulation where one DNA molecule is attached to another. The most important tools for splicing genes are the restriction endonuclease enzymes which cut DNA at specific sites and Produce specific DNA fragments which can be joined to some. Other DNA are techniques available for breaking a DNA molecule into shorter fragments.

Two such important techniques for splicing genes are the fragmentation of DNA by cleaving with restriction enzymes or by the synthesis of complementary DNA. The DNA fragments so isolated or synthesized are subsequently separated and then joined together if the ends are Complementary.

Gene Cloning:

Gene cloning is also referred to as DNA cloning or molecular cloning. In simple words it is the introduction of recombinant DNA molecule into a host cell which is then multiplied to produce the clones of rec DNA. However, often this term i.e., gene cloning is used as a synonym to rec DNA technology or genetic engineering.

Taq Polymerase:

It is a DNA polymerase enzyme which has an important role in PCR (Polymerase Chain Reaction).

It is a DNA polymerase-I type of enzyme which is isolated from the bacteria Thermus aquaticus which lives in hot springs. Taq polymerase enzyme is a thermo stable enzyme and it can withstand even the high temperature used for denaturation of DNA in PCR I hat is why, it is the suitable enzyme for polymerisation of DNA during PCR.

Human Genome Project:

Human Genome project (HGP) is an ambitious plan which is administered by National Institute of Health and Department of Energy, U.S.A. The idea behind HGP is to map and sequence all the genes found in human genome.

The main goal of the HGP is to obtain a complete knowledge of the structure, function, organisation and sequence of human DNA Thus the ultimate aim of HGP is to know the sequence of bases of each gene of a human and to apply this knowledge for benefits of mankind. The development of rec DNA technology specially the use of restriction enzymes has provided an extra boost to Human Genome Project.

Risks of Genetic Engineering:

It is true that biotechnology and genetic engineering have immense applications in almost all the areas related to the betterment of humanity.

But there are certain risks/harms related to it also It has been found that biotechnological processes may also have adverse effects in several areas. A destructive mind can use the biotechnological tools and techniques like genetic engineering for production of new arms race or biological warfare, etc.

Producing new and superior breeds of plants and animals can pose a danger to biodiversity, as only superior breeds would be used and others would be excluded. It is not wrong to say that if recDNA technology is not handled with a caution, it may prove to be disastrous.

There are a few confirmed cases showing the negative aspects of genetic engineering, these are listed below:

i. Genetically engineered human growth hormone (HGH) was found to cause leukemia in children on consumption, so, it was banned for sale in U.S.

ii. U.S. scientists reported the production of a super- AIDS virus which was formed when the ordinary AIDS virus was combined with the mice virus. It is believed to be more hazardous and can be transmitted even by air.

iii. A genetically engineered soybean manufactured by a British company was banned as it caused allergies in some persons on consumption.

iv. The transgenic maize manufactured by a Swiss company Ciba-Geigy was denied permission for sale because there was a fear that the antibiotic resistance gene present in it might go into the bacteria.

Several risks associated to the genetic engineering can be summarized as follows:

1. Hazardous toxins can be produced by genetic engineering of several organism like botulinum toxin, neurotoxins, aflatoxins etc.. can be used as biological weapons.

2. Advancements in biotechnology has also aided the terrorists to produce potent biological warfare agents. The genetically engineered microbes causing severe diseases like E.coli, Haemophilus influenza, etc., can be used for the same.

3. Large scale release of genetically modified plants may disturb the ecological and environmental equilibrium.

4. Introduction of superior genetically engineered varieties are replacing the wild type varieties and are causing a considerable loss to biodiversity.

5. During gene transfer process, the antibiotic resistance marker genes might get introduced to the bacteria which are pathogenic to humans. This may pose a great difficulty to get rid of this bacteria.

6. Gene drug preparation and Gene therapy approaches involve the introduction of genes into the target cells. There is a fear that incorrect integration of gene into target cells may cause problems by inactivating the essential genes.

7. Careless handling of tools of genetic engineering may result into the escape of organisms carrying recDNA molecule from the laboratory into the natural environment. This would be extremely harmful if it is involving a pathogenic gene.

8. Bacillus thuringiensis, baculoviruses, etc. are modified genetically to produce potent pesticides. If such GMOs attack the non-specific targets, they would result into disastrous consequences.

9. During the process of genetic modification the insertion of foreign gene into incorrect site in the host genome may result into the progeny with deformities.

10. GEM may disturb the ecosystem in which it is released by its rapid rate multiplication which may affect the native microbes of that ecosystem.

11. There is a likelihood of the transgene (like insect resistance, herbicide resistance, etc.) to be transferred from GMP to a related sexually compatible weed species. In such case the weed would become more persistent and it would be difficult to control it.

12. Some transgenic plants may pose threats to the human health by production of toxic and/or allergic metabolites. When consumed, such plant products cause allergy and/or infection to the human consumers.

From the above discussion it is clear that the field of biotechnology especially the genetic engineering is a double edged sword. The advances in genetic engineering are of immense help for humanity but if mishandled, their prospects are quite frightening.