Inclusion of double helix structural oligonucleotide (STexS) results in an enhance of SNP specificity in PCR

Jae Jong Kim,In Kyung Park,Hyoung-Min Park,Si-Kyu Lim,A Young Kyoung,Byoung Chul Park

doi:10.1038/s41598-021-98610-8

Abstract

Genetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers. Among of the methods developed for efficient detection of such SNP, polymerase chain reaction (PCR) methods are widely used worldwide for its cost and viable advantages. However, the technique to discriminate small amounts of SNP mixed in abundant normal DNA is incomplete due to intrinsic technical problems of PCR such as amplification occurring even in 3’mismatched cases because of high enzyme activity of DNA polymerases. To overcome the issue, specifically designed PCR platform, STexS (SNP typing with excellent specificity) using double stranded oligonucleotides was implemented as a means to emphasize the amplification of SNP templates by decreasing unwanted amplification of 3’mismatched DNA copies. In this study, the results indicate several EGFR mutations were easily detected specifically utilizing the STexS platform. Further trials show the novel method works effectively to discriminate mutations in not only general allele specific (AS)-PCRs, but also amplification refractory mutation system (ARMS)-PCR. The STexS platform will give aid in PCRs targeting potential SNPs or genetically mutated biomarkers in human clinical samples.

Highlights

Genetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers
The typical allele specific polymerase chain reaction (PCR) (AS-PCR) proceeds even when a mismatch occurs in the 3’ end of a primer. This is due to DNA polymerase reactions carrying on the dNTP even when a mismatch happens, which after the cycle converts to a normal match that continues amplifying
According to the kinetics of PCR in the report mentioned above, polymerization is continued without DNA polymerase detach due to high Kcat[1] of 3′ matched complex (DNAP·P/T1), but DNA polymerase will repeatedly be detached and attached in the event of discontinuous polymerization due to the low K cat[2] of 3′ mismatched complex (DNAP·P/T2) (Sup Fig. 1c and d)

Summary

Introduction

Genetic mutations such as single nucleotide polymorphisms (SNP) are known as one of the most common forms which related to various genetic disorders and cancers. The STexS platform will give aid in PCRs targeting potential SNPs or genetically mutated biomarkers in human clinical samples. The investigation of genetic variations which involves in genetic disorders and cancers is essential for predicting cures, establishing treatment methods, and observing prognosis and relapse. Identifying these variations are critical for human welfare, and for the selection and breeding of species and origin verification in agriculture[1].

Methods

Results

Conclusion