Recombinant DNA Technology

Recombinant DNA technology

Genetic engineering, a kind of biotechnology, is the latest branch in applied genetics dealing the alteration of the genetic make up of cells by deliberate and artificial means. Genetic engineering involves transfer or replacement of genes, so also known as recombination DNA technology or gene splicing.

Tools of genetic engineering : Two enzymes used in genetic engineering are restriction endonuclease and ligases. R.E. is used to cut the plasmid as well as the foreign DNA molecules of specific points while ligase is used to seal gaps or to join bits of DNA.

Steps of recombinant DNA technology

(1) Isolating a useful DNA segment from the donor organism.

(2) Splicing it into a suitable vector under conditions to ensure that each vector receives no more than one DNA fragment.

(3) Producing of multiple copies of his recombinant DNA.

(4) Inserting this altered DNA into a recipient organism.

(5) Screening of the transformed cells.

Vectors : Vector in genetic engineering is usually a DNA segment used as a carrier for transferring selected DNA into living cells. Which are as follows :

(1) Plasmid : Plasmid are extrachromosomal, closed circular double stranded molecules of DNA present in most eukaryotes. All plasmid carry replicons pieces of DNA that have the genetic information required to replicate. Plasmid pBR 322 was one of the first widely used cloning vectors, it contain both ampicillin and tetracycline resistance genes.

(2) Phage : It is constructed from the phage l chromosomes and acts as bacteriophage cloning vectors.

(3) Cosmid : The hybrids between plasmid and the phage l chromosome give rise to cosmid vectors.

Beside all these there are artificial chromosomes like

BACs (Bacterial Artificial chromosomes)

YACs (Yeast Artificial chromosomes)

MACs (Mammalian Artificial chromosomes) are very efficient vectors for eukaryotic gene transfers.

Natural genetic engineer : When as gene transfer occurs without human effort, the bacterium is known as “natural genetic engineer” of plants. e.g., A soil inhabiting, plant pathogenic bacterium, Agrobacterium tumefaciens.

Application of recombinant DNA technology : The technique of recombinant DNA can be employed in the following ways :

(1) It can be used to elucidate molecular events in the biological process such as cellular differentiation and ageing. The same can be used for making gene maps with precision.

(2) In biochemical and pharmaceutical industry, by engineering genes, useful chemical compounds can be produced cheaply and efficiently which is shown in table.

(3) Production of transgenic plants.

(4) Production of genetically modified microorganisms.

Genetic engineering in plants

The main steps in plant genetic engineering are given below :

(1) Agronomically important gene is identified and isolated.

(2) Plasmid is isolated from the bacterium, Agrobacterium tumefaciens.

(3) Plant DNA containing the gene of interest in integrated into the T DNA of the plasmid by using restriction endonuclease and ligase enzymes.

(4) Recombinant plasmid is introduced into the cultured plant cells.

(5) T DNA integrates into the plant cells chromosomes DNA.

(6) As the plant cells divide, each daughter cell receives a copy of T DNA and the gene of interest it carries.

(7) The cells give rise to a plantlet, which, when transferred into soil, grows into a new plant that may express the new gene.   

Cloning

Cloning is the process of producing many identical organisms or clones. In this process nucleus of ovum (n) is removed and replaced by nucleus of diploid cell of same organism. Now the egg with 2n nucleus is transferred to the uterus of mother to have normal pregnancy and delivers clone of itself.

Examples of organism cloning

(1) Cloning of sheep was done by Dr. Ian Wilmut (1995) of Roslin Institute, Edinberg U.K. and normal healthy lamb (DOLLY) was born in Feb, 1996. This lamb was exactly similar to her mother.

(2) The first cloned calves George and Charlie were born in January 1998.

(3) ANDI was the world’s first genetically altered primate produced by inserting a jelly fish gene into the embryo of a rhesus monkey.

(4) Scientist at Scotland cloned POLLY and MOLLY. Unlike Dolly, polly and molly were transgenic (they carried human protein gene) polly and molly were born in july 1997.

(5) Brigitte Boissliar, a 46-year old French chemist announced the creation of the world’s first cloned human baby nicknamed 𠇎ve” (December 2002).

Few examples of applications of plant cloning in genetic engineering are given below where desired DNA has been introduced in plant genome for various purposes :

Gene libraries

A gene library is a collection of gene clones that contains all the DNA present in some source. If the original source of the DNA was original DNA from a living organism, then the library seek to include clones of all that DNA, it is called a genomic gene library. Gene libraries can also be created by using RNA.  

cDNA

If a gene library is created by enzymatic copying of RNA by reverse transcriptase (RNA-dependent DNA polymerase), it would be called c-DNA library. c-DNA stands for complimentary DNA or copy DNA. c-DNA is made to use PCR to amplify an RNA. PCR does not work on RNA, so one can copy it to DNA using reverse transcriptase and then PCR amplify the c-DNA this is called RT-PCR (reverse transcriptase PCR).

Gene bank

A gene bank is repository of clones of known DNA fragments, genes, gene maps, seeds, spores, frozen sperms or eggs or embryos. These are stored for possible use in genetic engineering and breeding experiment where species have become extinct.

DNA finger printing

Alec Jeffreys et al (1985) developed the procedure of genetic analysis and forensic medicine, called DNA finger printing. It is individual specific DNA identification which is made possible by the finding that no two people are likely to have the same number of copies of repetitive DNA sequences of the regions. It is also known as DNA profiling. The chromosomes of every human cell contain scattered through their DNA short, highly repeated 15 nucleotide segments called “mini-satellites” or variable-number Tandem Repeat (VNTR).

Applications of DNA fingerprinting

This technique is now used to :

(1) Identify criminals in forensic laboratories.

(2) Settle paternity disputes.

(3) Verify whether a hopeful immigrant is, as he or she claims, really a close relative of already an established resident.

(4) Identify racial groups to rewrite biological evolution.

Gene therapy

The use of bioengineered cells or other biotechnology techniques to treat human genetic disorders is known as gene therapy. Gene therapy is the transfer of normal genes into body cells to correct a genetic defect. It can be used to treat genetic diseases like sickle-cell anaemia and Severe Combined Immuno Deficiency (SCID). It (SCID) is caused by a defect in the gene for the enzyme adenosine deaminase (ADA). SCID patients have no functioning T lymphocytes and one treated with the injections of their white blood cells that have been engineered to carry the normal ADA alleles.

Transgenics

A gene that has been introduced into a cell or organism is called a transgene (for transferred gene) to distinguish it from endogenous genes. The animal carrying the introduced foreign gene is said to be transgenic animal and the possessor called Genetically Modified Organisms (GMOs). Most of the transgenic animals studied to date were produced by microinjection of DNA into fertilized eggs. Prior to microinjection, the eggs are surgically removed from female parent and fertilized in vitro then DNA is microinjected into the male pronucleus of the fertilized egg through a very fine-tipped glass needle. The integration of injected DNA molecules appears to occur at random sites in the genome.

The first transgenic animal produced was the ‘supermouse’ by the incorporation of the gene for human growth hormone by Richard Palmiter and Ralph Brinster in 1981.