Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers

Juny Shin, Cheulhee Jung

Research output: Contribution to journalArticlepeer-review


Early detection of rare mutations through liquid biopsy can provide real-time information related to cancer diagnosis, prognosis, and treatment outcomes. Cell-free DNA samples used in liquid biopsies contain single-nucleotide variants (SNVs) with a variant allele frequency (VAF) of approximately ≤1%. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard of sequencing using liquid samples, generating amplicons from samples containing mutations with 0.001–0.005% VAF; however, it requires expensive equipment and time-consuming protocols. Therefore, various PCR methods for discriminating SNVs have been developed; nonetheless, non-specific amplification cannot be avoided even in the absence of mutations, which hampers the accurate diagnosis of SNVs. In this study, we introduce single-nucleotide variant on–off discrimination–PCR (Soo-PCR), a highly accurate and practical method that uses a 3′-end tailing primer for the on–off discrimination of low-abundance mutant-type targets, including SNVs. Soo-PCR minimizes the chance of incorrect judgments owing to its high discriminating power. Cancer markers, such as KRAS G12D, EGFR L858R, and EGFR T790M mutations, containing 0.1% VAF, were clearly detected in under 2 h with a high reliability comparable with that of ddPCR. This new method serves as a practical approach to accurately detect and evaluate low-abundance mutations in a user-friendly manner.

Original languageEnglish
Article number380
Issue number3
Publication statusPublished - 2023 Mar


  • liquid biopsy
  • low-abundance mutation
  • on–off discrimination
  • polymerase chain reaction
  • single-nucleotide variant

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biomedical Engineering
  • Instrumentation
  • Engineering (miscellaneous)
  • Clinical Biochemistry


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