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Isolation of Genes Coding for Specific Proteins - Isolation of genes for specific proteins became possible only after the discovery of reverse transcriptase enzyme in 1970. This enzyme can be easily used for the synthesis of copy DNA or complementary DNA (cDNA) from mRNA. This complementary DNA can then be used for the isolation of the corresponding gene from genomic DNA. It is, therefore, obvious that for the isolation of a specific gene, techniques should first be available for the isolation of specific mRNA:

For this purpose, antibodies are produced against a specific protein for which the gene is to be isolated.

Therefore, isolation of a gene coding for a specific protein involves the following basic steps;

(i) purification of the protein product of the gene;

(ii)Production of antibodies against this protein product by immunizing animals like rabbit or mouse;

(iii) precipitation of polysomes engaged in synthesizing specific' protein with the help of the antibody raised in step.

(iv) isolation and purification of mRNA from the Polysome fraction;

(v) using mRNA for synthesizing cDNA with the help of reverse transcriptase;

(vi) cloning of cDNA thus synthesized for preparation of a cDNA library;

(vii) immunological and electrophoretic analysis of the translation products of cDNA clones to identify specific cDNA clone meant for the specific protein in mind;

(viii) use of the specific cDNA probes selected in step

(ix) for the identification and isolation of the gene from genomic DNA through screening a complete or partial genomic library.

In step (vi) above for preparation of cDNA library, cDNA may be cloned in expression vectors (vectors in which gene can be expressed to the presence of promoter sequences for RNA polymerase enzyme) like λgt 11, which can accept gene insertions into β galactosidase gene. The chimeric vector will then produce hybrid protein if correctly expressed.

The hybrid protein can be identified using antibody as above. Plaques belonging to the chimeric vector carrying the desired gene can be identified by the reaction of antibody attached to a radioactve protein. This procedure was used to clone a number of genes including a gene regulating zein synthesis in maize.

The cDNA clones may also be used directly for gene manipulation and transformation experiment. However, when cDNA clone is used, it should be described as synthesis of gene rather than as isolation of gene, since cDNA has been artificially synthesized, and not isolated from the organism. The above technique for isolation of gene has now been successfully utilized both in plants and animals.

However, the first genes isolated were those which existed in multiple copies and were expressed it high levels in specific tissues, e.g. ovalbumin gene in chicks, globin and immunoglobulin genes in mouse, genes for storage proteins in cereals and legumes, amylase genes in barley, lactin genes in some legumes, etc.Isolation of genes which are tissue specific in expression.
 

 
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