Disruptors, a new class of oligonucleotide reagents, significantly improved PCR performance on templates containing stable intramolecular secondary structures

Liu, Zhaocheng; Zhao, Chen; Zhao, Guodong; Xiong, Shangmin; Ma, Yong; Zheng, Minxue

doi:10.1016/j.ab.2021.114169

Cited by 3 publications

(1 citation statement)

References 43 publications

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“…To solve the dilemma caused by blocker addition in BDA, we contrived a novel multiplex method to enrich low-abundance variants without using any additional blocker reagent. The hairpin is a critical issue affecting the hybridization of DNA. − Our previous work has shown that PCR would be impeded by thermodynamically stable hairpins due to cleavage of structured templates by Taq polymerase. , Inspired by these investigations, we assumed that if artificial hairpins were introduced into templates engineered to compete with primers in PCR, hairpins would have varied stability and consequently, primer annealing would have varied efficiency due to single-nucleotide variants in the wild and mutant templates. In this way, rare variants could be amplified specifically and discriminated from wild-type DNA coupled with qPCR and Sanger sequencing.…”

Section: Introductionmentioning

confidence: 99%

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

Liu,

Zhang,

Jiang

et al. 2023

Anal. Chem.

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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.

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