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.