Andrew S. Wiecek, 02/23/2011:
According to the WPI group, nested PCR of cultured samples provided the best results for XMRV detection. Capable of amplifying single molecules within a large sample, it is used when targeted DNA templates are in extremely low concentrations. So-called for the way it “nests” two runs using two sets of primers, the product of the first PCR run becomes a template for the second. The technique’s only limitation is the amount of template DNA in the sample.
To increase the sensitivity of their nested PCR protocol, Mikovits turned to Max Pfost, a graduate student in her lab. Pfost researched PCR optimization and began modifying the protocol’s magnesium concentrations and annealing conditions, and choose primers for increased sensitivity rather than specificity.
Mikovits allegorizes from what she calls “the HIV days,” when it was first discovered that multiple low–copy number HIV strains existed.
“If you are looking for a low–copy number family member, you really have to optimize the magnesium and base everything on the annealing temperatures." Otherwise, she says, researchers using nested PCR would very likely overlook XMRV in their samples.
“A lot of people are just not taking the time to optimize their PCR,” says Pfost. Read more>>