The End Process Of Screening For The Desired Clones
Nucleic acid hybridization is the most commonly used method for the screening because it is rapid and can be applied to a very large numbers of clones. They can also be used to identify the clones that are not of full-length. Often radioactive-RNA probes are used.
The PCR is widely used to isolate specific DNA sequences from uncloned genomic DNA or cDNA. It is also a useful technique for library screening. It is possible to identify any clone by PCR but only if there is sufficient information about its sequence to make suitable primers. To isolate a specific clone, PCR is carried out with gene-specific primers that flank a unique sequence in the target.
Immunological screening involves the use of antibodies that specifically recognize antigenic determinants on the polypeptide synthesized by a target clone. It can be applied to any protein for which an antibody is available and hence is one of the best screening methods of clones.
d) Protein-protein interaction
DNA-binding proteins are used for the screening of clones. The screening is carried out, by incubating with a radio-labeled double-stranded DNA oligonucleotide probe, containing the recognition sequence for the target DNA-binding protein.
e) Protein-ligand interaction
A wide range of ligands can be used to identify polypeptides that specifically bind certain molecules. However this method has a very low sensitivity hence it is not preferred.
f) Functional complementation
It is a very powerful method of expression cloning, as the non-viable mutant cells carrying the clones of interest can be positively selected under a particular growth conditions, allowing the corresponding clones to be isolated.
g) Gain of function
Sometimes it is possible to identify clones on the basis of the gain of function they confer to the host cell. This function may be a selectable phenotype in some cases, and therefore allow the cells containing the corresponding clone to be positively selected.
Four different types of enzymes are used viz. nucleases, ligases, polymerases, and certain DNA modifying enzymes.
- For the production and isolation of the DNA fragment one of the following methods may be used:
- For the insertion of the gene into a suitable vector either of these methods are used:
- For further introduction of the recombinant DNA molecule into the host cell one of these methods is suitable:
- The end process of screening for the desired clones is carried out in one of the following manner:
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