Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer

Abstract

PCR assays are the most rapid, most sensitive, and least expen- sive way to assess the abundance of mutant DNA fragments present in liquid biopsies. “SuperSelective” PCR primers, due to their unique design, are extraordinarily specific, able to selectively initiate the synthesis of amplicons on ten mutant DNA fragments in the presence of 1,000,000 wild-type DNA fragments. Sets of SuperSe- lective primers, each possessing unique 5′-tag sequences, enable the amplicons generated from each mutant to be distinguished by differently colored molecular beacon probes. And the inclusion of primers for a wild-type reference gene fragment, enables the abundance of each type of mutant DNA fragment to be assessed by determining the difference between its threshold value and the threshold value of the reference gene.
http://dx.doi.org/10.1016/j.bdq.2017.02.079