Journal Article

DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA

Tongbo Wu, Wei Chen, Ziyu Yang, Haocheng Tan, Jiayu Wang, Xianjin Xiao, Mengyuan Li and Meiping Zhao

in Nucleic Acids Research

Volume 46, issue 4, pages e24-e24
Published in print February 2018 | ISSN: 0305-1048
Published online November 2017 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkx1218
DNA terminal structure-mediated enzymatic reaction for ultra-sensitive discrimination of single nucleotide variations in circulating cell-free DNA

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Abstract

Sensitive detection of the single nucleotide variants in cell-free DNA (cfDNA) may provide great opportunity for minimally invasive diagnosis and prognosis of cancer and other related diseases. Here, we demonstrate a facile new strategy for quantitative measurement of cfDNA mutations at low abundance in the cancer patients’ plasma samples. The method takes advantage of a novel property of lambda exonuclease which effectively digests a 5′-fluorophore modified dsDNA with a 2-nt overhang structure and sensitively responds to the presence of mismatched base pairs in the duplex. It achieves a limit of detection as low as 0.02% (percentage of the mutant type) for BRAFV600E mutation, NRASQ61R mutation and three types of EGFR mutations (G719S, T790M and L858R). The method enabled identification of BRAFV600E and EGFRL858R mutations in the plasma of different cancer patients within only 3.5 h. Moreover, the terminal structure-dependent reaction greatly simplifies the probe design and reduces the cost, and the assay only requires a regular real-time PCR machine. This new method may serve as a practical tool for quantitative measurement of low-abundance mutations in clinical samples for providing genetic mutation information with prognostic or therapeutic implications.

Journal Article.  7483 words.  Illustrated.

Subjects: Research Methods in Life Sciences

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