Abstract

Multiplex Real-time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

Fred Kramer, Professor, New Jersey Medical School Rutgers University

Real-time multiplex PCR assays are potentially the most rapid, most sensitive, and least expensive way to assess the abundance of mutant DNA fragments present in liquid biopsies; provided that a way is found to selectively amplify these rare mutant fragments without amplifying the abundant wild-type fragments; and provided that the amplicons generated from different mutants are prevented from forming heteroduplexes that interfere with exponential amplification, obscuring the threshold values of the rarer mutants. “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, even though the only difference between the mutant and the wild-type is a single-nucleotide polymorphism. Moreover, each SuperSelective primer specific for a particular mutation possesses a unique 5’ tag sequence that is incorporated into the resulting amplicons and detected in real-time by differently colored molecular beacon probes. Each SuperSelective primer specific for a particular mutation also possesses an additional unique “bridge” sequence that assures that each primer only copies its intended amplicon, and that creates a single-stranded bubble in heteroduplexes that enables the rarest amplicons to be independently exponentially amplified. And finally, the inclusion of primers for a wild-type reference gene fragment, enables the abundance of each type of mutant DNA fragment to be assessed (without measuring the amount of DNA in the sample) by determining the difference between its threshold value and the threshold value of the reference gene.