Multiplex SuperSelective PCR Assays for the Detection and Quantitation of Rare Somatic Mutations in Liquid Biopsies.

SuperSelective primers, by virtue of their unique design, enable the simultaneous identification and quantitation of inherited reference genes and rare somatic mutations in routine multiplex PCR assays, while virtually eliminating signals from abundant wild-type sequences closely related to the target mutations. These assays are sensitive, specific, rapid, and low cost, and can be performed in widely available spectrofluorometric thermal cyclers. Herein, we provide examples of SuperSelective PCR assays that target eight different somatic EGFR mutations, irrespective of whether they occur in the same codon, occur at separate sites within the same exon, or involve deletions. In addition, we provide examples of SuperSelective PCR assays that detect specific EGFR mutations in circulating tumor DNA present in the plasma of liquid biopsies obtained from patients with non-small-cell lung cancer. The results suggest that multiplex SuperSelective PCR assays may enable the choice, and subsequent modification, of effective targeted therapies for the treatment of an individual's cancer, utilizing frequent noninvasive liquid biopsies.

Suppression of Wild-Type Amplification by Selectivity Enhancing Agents in PCR Assays that Utilize SuperSelective Primers for the Detection of Rare Somatic Mutations.

In PCR assays designed to detect rare somatic mutations, SuperSelective primers, by virtue of their short 3'-foot sequences, selectively initiate synthesis on mutant DNA target fragments, while suppressing the synthesis of related wild-type fragments, and the resulting threshold cycle reflects the quantity of mutant targets present. However, when there are ≤10 mutant target fragments in a sample, the threshold cycle that is observed occurs so late that it can be confused with the threshold cycle that arises from samples that contain only abundant related wild-type fragments. We report here that the inclusion of the selectivity enhancing agents tetramethylammonium chloride or bis-tetramethylammonium oxalate in SuperSelective PCR assays substantially suppresses the amplification of related wild-type fragments. As a result of this selective suppression, assay sensitivity is increased to such an extent that multiplex PCR assays can be performed in which it is highly unlikely that there will be a false-positive or false-negative result. This advance provides a foundation for the development of rapid, low-cost, multiplex PCR assays for noninvasively assessing the presence of relevant mutations in cancer patients, thereby enabling individually appropriate therapy.