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

J Mol Diagn. 2018 Jul;20(4):415-427. doi: 10.1016/j.jmoldx.2018.03.004. Epub 2018 Apr 24.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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


  • DNA Primers
  • KRAS protein, human
  • Quaternary Ammonium Compounds
  • DNA
  • Oxalic Acid
  • DNA-Directed DNA Polymerase
  • Proto-Oncogene Proteins p21(ras)
  • tetramethylammonium