Highly Sensitive Enrichment of Low-Frequency Variants by Hairpin Competition Amplification

Gene mutations are inevitably accumulated in cells ofthe humanbody. It is of great significance to detect mutations at the earliestpossible time in physiological and pathological processes. However,genotyping low-copy tumor DNA (ctDNA) in patients is challenging dueto abundant wild DNA backgrounds. One novel strategy to enrich raremutations at low variant allele fractions (VAFs) with quantitativepolymerase chain reaction (qPCR) and Sanger sequencing was contrivedby introducing artificial hairpins into amplicons to compete withprimers, coined as the hairpin competition amplification (HCA) system.The influence imposed by artificial hairpins on primer-binding ina high-temperature PCR system was investigated for the first timein this work, paving the way for the optimization of HCA. HCA differsfrom the previously reported work in which hairpins are formed toinhibit extension of wild-type DNA using 5-exonuclease-negative polymerase,where the readout is dependent on melting curve analysis after asymmetricPCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enrichedmutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstratedadvantages in multiplex reaction and temperature robustness. In profilinggene status from 12 lung cancer ctDNA samples and 16 thyroid cancerFNA DNA samples, HCA demonstrated a 100% concordance rate comparedto ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing canenrich low-abundance variants with high sensitivity and temperaturerobustness, presenting a novel and effective tool for precision diagnosisand treatment of rare variant diseases.