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

• Published in: The Journal of molecular diagnostics : JMD Vol. 20; no. 4; pp. 415 - 427
• Main Authors: Vargas, Diana Y., Marras, Salvatore A.E., Tyagi, Sanjay, Kramer, Fred R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2018
• Subjects: Alleles; Base Sequence; DNA - blood; DNA - genetics; DNA - isolation & purification; DNA Mutational Analysis - methods; DNA Primers - metabolism; DNA-Directed DNA Polymerase - metabolism; Humans; Multiplex Polymerase Chain Reaction - methods; Mutation - genetics; Oxalic Acid - chemistry; Proto-Oncogene Proteins p21(ras) - genetics; Quaternary Ammonium Compounds - chemistry; Reproducibility of Results; Suppression, Genetic
• ISSN: 1525-1578; 1943-7811
• DOI: 10.1016/j.jmoldx.2018.03.004

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.