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

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.

SUBMITTER: Wu T 

PROVIDER: S-EPMC5829738 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature