Site-specific insertion of endonuclease recognition sites into amplicons to improve post-PCR analysis sensitivity of gene mutation

Abstract

Precise detection of low-frequency gene mutations surrounded by excess wild-type DNA is important in many aspects of medical fields. Most hybridization-based methods for high-resolution mutant allele analysis are hindered by competition of the complementary strand with single-strand probes for the target strand. Here, we demonstrate that site-specific insertion of endonuclease recognition sites into amplicons allows post-PCR generation of short dsDNA or ssDNA, whereby improves the sensitivity of both melting temperature analysis (MTA) and end-point detection following up. Using a three-staged PCR protocol, enrichment of target gene and incorporation of specific restriction sites in amplicons were ensued with hardly any loss in amplification efficiency and specificity. It enables simultaneous discrimination among a panel of totally 11 EGFR 19 exon deletion mutations via MTA after post-PCR digestion by either FokI only or cooperated with CRISPR-Cas12a, using SYBR green I. By replacement of one double-strand cleavage site with a nickase binding domain post-PCR generation of ssDNA of interest via strand displacement amplification (termed as iSDA) is realized. Our preliminary investigation shows that iSDA permits analysis of single nucleotide variants down to 0.1% allelic-frequency using end-point detection. Given the good compatibility with the majority of mutant-enrich PCR methods, we envision it would advance the current gene profiling technologies to a large extent.