Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Hybridation vs Normal PCR - (Oct/26/2005 )

When you want to get a new sequence which has not been published yet, you use among other methods hybridation, am I right? But when the sequence has already been published, then what is the interest of using this hybridating process? I've seen a few papers which use this method. Isn't it better and easier using standard PCR with primers according to the published sequence and then normal clonning?

Thanks for your comments.


The answer is somewhat yes. If you have a partial sequence or are trying to pull out a gene by regions of homology, many people probe a library by hybridizing a probe to the genomic sequences. An alternative method is degenerate PCR which is more sensitive. Once you have the known sequence, PCR is a much easier and sensitivive assay.


Plus, there are circumstances under which a Southern blot would detect a homologous sequence, but PCR using primers synthesized from the published sequence would fail.

Consider the consequences of a 30-bp change in a 1 kb sequence, for example. The target sequence is still 97% identical to the reference sequence but, if you happened to pick primers that relied on one or more of the altered bases (especially at the 3' end of the primer), the PCR could fail. The Southern blot, however, would easily detect the presence of this gene...