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 language | English |
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Article number | 380 |
Journal | Biosensors |
Volume | 13 |
Issue number | 3 |
DOIs | |
Publication status | Published - 2023 Mar |
Bibliographical note
Funding Information:This work was supported by the Technology Innovation Program and the Korea Industrial Complex Corporation Project funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) (grant numbers 20009356 and IRGJ2203); a National Research Foundation of Korea grant funded by the Korean government (MSIT) (grant number 2021R1C1C1004147); and the Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science & ICT (grant number 2022M3A9B6082670).
Funding Information:
We gratefully acknowledge the support of Korea University for this study.
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