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

Abstract

Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and pathological processes. However, genotyping low-copy tumor DNA (ctDNA) in patients is challenging due to abundant wild DNA backgrounds. One novel strategy to enrich rare mutations at low variant allele fractions (VAFs) with quantitative polymerase chain reaction (qPCR) and Sanger sequencing was contrived by introducing artificial hairpins into amplicons to compete with primers, coined as the hairpin competition amplification (HCA) system. The influence imposed by artificial hairpins on primer-binding in a high-temperature PCR system was investigated for the first time in this work, paving the way for the optimization of HCA. HCA differs from the previously reported work in which hairpins are formed to inhibit extension of wild-type DNA using 5-exonuclease-negative polymerase, where the readout is dependent on melting curve analysis after asymmetric PCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enriched mutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstrated advantages in multiplex reaction and temperature robustness. In profiling gene status from 12 lung cancer ctDNA samples and 16 thyroid cancer FNA DNA samples, HCA demonstrated a 100% concordance rate compared to ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing can enrich low-abundance variants with high sensitivity and temperature robustness, presenting a novel and effective tool for precision diagnosis and treatment of rare variant diseases.

Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration

“…DMT was specifically amplified within 30 min using the forward primer “S” and reverse primer “A” (Figure S3A,B). The PCR products were further amplified via asymmetric PCR, employing primer S at a higher concentration than primer A to yield abundant single strands. , Under the optimized conditions with a concentration ratio of primer S to primer A of 20:1, the sensitivity of various gene templates was evaluated. As shown in Figure A,B, FAM fluorescence increased with the rising concentration of T790M, and ROX fluorescence similarly increased in the presence of C797S.…”

“…The PCR products were further amplified via asymmetric PCR, employing primer S at a higher concentration than primer A to yield abundant single strands. 27,28 Under the optimized conditions with a concentration ratio of primer S to primer A of 20:1, the sensitivity of various gene templates was evaluated. As shown in Figure 4A,B form Trans templates, an enhanced FAM and ROX fluorescence signal from the 4WJ probe was observed at Trans concentrations above 2 × 10 3 copies/μL (Figure 4C).…”