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Single‐molecule detection of cancer mutations using a novel PCR‐LDR‐qPCR assay
Author(s) -
Ruiz Cristian,
Huang Jianmin,
Giardina Sarah F.,
Feinberg Philip B.,
Mirza Aashiq H.,
Bacolod Manny D.,
Soper Steven A.,
Barany Francis
Publication year - 2020
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.23987
Subject(s) - kras , biology , microbiology and biotechnology , primer (cosmetics) , cold pcr , oligonucleotide , point mutation , dna , cancer , wild type , mutant , mutation , polymerase chain reaction , cancer research , genetics , gene , chemistry , organic chemistry
Detection of low‐abundance mutations in cell‐free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR‐LDR‐qPCR assay capable of detecting point mutations at a single‐molecule resolution in the presence of an excess of wild‐type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer‐binding regions as well as wild‐type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF :c.1799T>A (p.Val600Glu), TP53 :c.743G>A (p.Arg248Gln), KRAS :c.35G>C (p.Gly12Ala), KRAS :c.35G>T (p.Gly12Val), KRAS :c.35G>A (p.Gly12Asp), KRAS :c.34G>T (p.Gly12Cys), and KRAS :c.34G>A (p.Gly12Ser). The single‐well version of the assay detected 2–5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild‐type human genomic DNA (hgDNA) from buffy coat. A 12‐well (pixel) version of the assay was capable of single‐molecule detection of the aforementioned mutations at TP53 , BRAF , and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000–2,250 GE of wild‐type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.