Recombinant DNA Technology and Gene Cloning

Biotechnology in Agriculture
The New Green Revolution
Nucleic Acids
Protein Synthesis
Southern and Northern Blot Techniques
Nucleic Acid Hybridization
Recombinant DNA Technology and Gene Cloning
Some Restriction Enzyme Sequences the Arrow Indicates Actual Clevage Site
Gene Exchange in Bacteria
The Agrobacterium Mediated Transfer of Genes
Procedures for Agrobacterium Mediated Transformations
Direct DNA Transfer Technologies
Antisense RNA Strategy
Frost Control Biotechnology
Virus Resistance in Transgenic Plants
Transformation of Sequences Related to Viral Capsid Protein
Resistance Conferred by Antisense RNA
Antibody Mediated Resistance

Recombinant DNA Technology and Gene Cloning

Recombinant DNA Technology and Gene Cloning
Man made nucleotide sequences tailored to carry desired traits, known as molecular probes have been extensively used in molecular biology studies. These nucleotide squences (probes) ought to be correct, pure and available in large amounts.

The probe must be labelled in some manner to help detection. These probes are prepared by recombinant DNA techniques using the common enteric bacterium Escherichia coli. This organism has been studied so often by molecular biologists that they are now aware of the implications of its genome containing about 4.2 million base pairs (bp).

Furthermore, the bacterium has extrachromosomal DNA molecules known as plasmids that can replicate autonomously independent of the nuclear DNA. These plasmids are closed single pieces of supercoiled DNA which can be stably inherited by daughter cells. They have the ability to replicate to high numbers (copies) within each bacterial cell.

The restriction endonucleases cleave DNA only to specific oligonucleotide sequences rather asymetrically leaving 'sticky' ends. The sticky ends remain complementary between any two DNA fragments sliced by the same restriction enzyme. This property makes it easy to insert or 'clone' an outsider or foreign gene to the E. coli plasmid provided it has been sliced by the same restriction enzyme.

A. Genomic DNA, B. Fragments after digestion with restriction enzyme	G. The probe Hybridizes with the target sequences that is identified by autoradiography

Separation by Gel Electrophoresis and Identification with a fluroscent dye such As Ethidium Bromide in a Southern Blotting Technique

The Sequence from the Southern blot is transferred an d bound to a solid Phase, Usually Nitrocellulose or Nylon Membrane

C. Separation by Gel Electrophoresis and Identification with a Fluroscent Dye such as ethidium bromide in a Southern blotting technique

E. Southern Blot is transferred and bound to a solid phase usually nitrocellulose or nylon membrane

The transformed plasmids known as 'vectors' can be amplified to a high copy number in a standard bacterial culture. To retrieve the inserted foreign DNA, the bacterial biomass from the culture medium is separated and lysed. The DNA content is purified and sliced by the same restriction enzyme that was earlier used for original cloning.

Plasmid vectors have been used to transfer DNA from one prokaryotic cell to another, from a prokaryote to an eukaryote and from an eukaryote to a prokarotic cell. The plasmid vectors have the limitations of cloning upto 5000 base pairs (bp) or 5 kilobases (kb). By developing and using bacteriophage lambda chromosomes, foreign DNA have been cloned upto 15 kb.

The ability to use vectors has been further enlarged by using features of both plasmids and bacteriophage lambda to the extent of 50 kb. Presently, specially constructed DNA fragments from yeast cells known as yeast artificial chromosomes (YAKs) are available that can be used to clone pieces of DNA upto 1 million bp.

One of the important steps in cloning a foreign gene is to obtain the desired gene in the absolute pure condition. This can be done by a traditional method beginning with the purified protein which the gene produces. Required antibodies are raised which will recognize and precipitate the protein when added to a cell extract from a tissue where the protein is actually synthesized.

This results in the precipitation of newly made polypeptides which are being elongated on the polyribosomes. However, the precipitate contains unwanted ribosomes mixed with the mRNA templates required for producing the protein in question. When these mRNA templates are purified from the mixture, a sequence of nucleotides complementary to the gene of interest can be obtained. By using a retroviral enzyme reverse transcriptase, which synthesizes DNA from a RNA template, a cDNA sequence from the mRNA sequence can be obtained.

By the addition of DNA polymerase the second strand of DNA can be replicated which results in the formation of a double stranded DNA copy of the gene of interest. This elaborate procedure is known as the polysome precipitation.

In recent years, polysome precipitation method has been replaced by 'DNA libraries'. A cDNA library is made up of all the actively transcribed genes of a tissue inserted into a population of bacterial cells. The bulk of mRNA preparation is reverse transcribed and inserted into plasmids in one lot, with the objective that every possible cDNA sequence will be carried by atleast one bacterial cell in the culture. This cDNA library has to be sorted out for a particular cDNA sequence of interest.

The bacterial culture containing the cDNA is spread out on agar plates and filter blot technique is used with a labelled oligonucleotide probe (1D-40 bp) to select a colony containing the foreign gene of interest. Presently, many biotechnological companies have developed automated instruments that can rapidly synthesize oligonucleotides of any sequence.

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