Combining E-ice-COLD-PCR and Pyrosequencing with Di-Base Addition (PDBA) Enables Sensitive Detection of Low-Abundance Mutations.

Autor: Pu D; Chongqing Key Laboratory of Big Data for Bio Intelligence, Chongqing University of Posts and Telecommunications, Chongqing, 400065, China., Chen H; Chongqing Key Laboratory of Big Data for Bio Intelligence, Chongqing University of Posts and Telecommunications, Chongqing, 400065, China., Fu W; Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China., Cui Y; Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China. cuiyouhongx@163.com., Shu K; Chongqing Key Laboratory of Big Data for Bio Intelligence, Chongqing University of Posts and Telecommunications, Chongqing, 400065, China. shukx@cqupt.edu.cn.
Jazyk: angličtina
Zdroj: Applied biochemistry and biotechnology [Appl Biochem Biotechnol] 2024 Jul; Vol. 196 (7), pp. 4049-4066. Date of Electronic Publication: 2023 Oct 21.
DOI: 10.1007/s12010-023-04718-0
Abstrakt: Detecting low-abundance mutations is of particular interest in the fields of biology and medical science. However, most currently available molecular assays have limited sensitivity for the detection of low-abundance mutations. Here, we established a platform for detecting low-level DNA mutations with high sensitivity and accuracy by combining enhanced-ice-COLD-PCR (E-ice-COLD-PCR) and pyrosequencing with di-base addition (PDBA). The PDBA assay was performed by selectively adding one di-base (AG, CT, AC, GT, AT, or GC) instead of one base (A, T, C, or G) into the reaction at a time during sequencing primer extension and thus enabling to increase the sequencing intensity. A specific E-ice-COLD-PCR/PDBA assay was developed for the detection of the most frequent BRAF V600E mutation to verify the feasibility of our method. E-ice-COLD-PCR/PDBA assay permitted the reliable detection of down to 0.007% of mutant alleles in a wild-type background. Furthermore, it required only a small amount of starting material (20 pg) to sensitively detect and identify low-abundance mutations, thus increasing the screening capabilities in limited DNA material. The E-ice-COLD-PCR/PDBA assay was applied in the current study to clinical formalin-fixed paraffin-embedded (FFPE) and plasma samples, and it enabled the detection of BRAF V600E mutations in samples that appeared as a wild type using PCR/conventional pyrosequencing (CP) and E-ice-COLD-PCR/CP. E-ice-COLD-PCR/PDBA assay is a rapid, cost-effective, and highly sensitive method that could improve the detection of low-abundance mutations in routine clinical use.
(© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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