In all cloning experiments, it is essential to verify that the correct clone has been obtained. The verification procedure is necessary to eliminate false positives arising as a result of ambiguities in the screening procedure, such as those due to unexpected antibody reactivities or highly degenerate oligonucleotide probes.
It is not uncommon to obtain false positives by antibody screening of bacterial expression libraries. Authentication should be performed using procedures that are independent of those used to obtain the clones in the first place.
Verification could include checks on the presence of hybridizing mRNA of the appropriate size and abundance in expressing cells, compared with non-expressing cells, or hybridization of the cloned gene with an independent oligonucleotide probe from a different peptide. Correct prediction of amino acid sequences lying outside the region used for design of the probe would be good evidence that the correct clone has been obtained, as would the ability of the cloned gene to transfer appropriate biological or antigenic activity in an expression system such as COS cells.
Occasionally, papers are published in which the wrong gene was cloned. In order to avoid embarrassment, it is wise to use several independent methods to check the authenticity of clones, and to treat the exercise of authentication just as seriously as the effort to isolate the original clone.
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