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

Juny Shin, Cheulhee Jung

    Research output: Contribution to journalArticlepeer-review

    2 Citations (Scopus)

    Abstract

    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
    JournalBiosensors
    Volume13
    Issue number3
    DOIs
    Publication statusPublished - 2023 Mar

    Bibliographical note

    Publisher Copyright:
    © 2023 by the authors.

    Keywords

    • 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

    Fingerprint

    Dive into the research topics of 'Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On–Off Discriminating Primers'. Together they form a unique fingerprint.

    Cite this