Thursday, October 27, 2011

Design of hybrid primers

Failures in PCR amplification is very common in a molecular lab as multiple factors can affect it (DNA quality,  parameter of the PCR program, use of non-specific primers etc.). So, we have to limit these issues as much as we can. One way is to design good primers. The problem is that often it is not possible to have 25bp of conserved region of interest for the studied taxon. A previous paper that focused on protein evolution got around this issue by showing that 3 to 4 conserved codons were enough to design a good primer (Rose et al. 1998). More recently, adapting this approach to nucleotide sequences, we developed a set of conserved (and efficient) COI primers for the echinoderm phylum (sea-urchins, sea-cucumbers, sea-lilies or crinoids, sea-stars and brittle-stars; Hoareau & Boissin 2010). I used the same approach to design the ND2 primers for the whole vertebrates. I think this is very worthy as the same set of primers can be used by everyone in the lab. See below the graph that shows a conserved 3' region for each primer.
Sometimes, it will not work if we cannot find a conserved region long enough to design the primers. But I suggest that if you have to design primers (other than microsatellites!) you should try this method. Soon enough, we will have only a small set of primers for all the species in the lab.


Alignment of ND2 sequences (+ flanking regions) of various vertebrates showing the regions where I designed the primers

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