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

作者全名："Song, Lin; Li, Junjie; Chen, Kena; Zuo, Chen; Wu, You; Bai, Dan; Zhao, Lin; Yang, Yujun; Liu, Chenggui; Xie, Guoming"

作者地址："[Song, Lin; Li, Junjie; Chen, Kena; Zuo, Chen; Wu, You; Bai, Dan; Yang, Yujun; Xie, Guoming] Chongqing Med Univ, Dept Lab Med, Minist Educ, Key Lab,Lab Med Diagnost, Chongqing, Peoples R China; [Zhao, Lin] Chongqing Med Univ, Dept Emergency & Crit Care Med, Affiliated Hosp 1, Chongqing, Peoples R China; [Liu, Chenggui] Univ Elect Sci & Technol China, Chengdu Womens & Childrens Cent Hosp, Sch Med, Dept Clin Lab, Chengdu, Peoples R China"

通信作者："Li, JJ; Xie, GM (通讯作者)，Chongqing Med Univ, Dept Lab Med, Minist Educ, Key Lab,Lab Med Diagnost, Chongqing, Peoples R China.; Liu, CG (通讯作者)，Univ Elect Sci & Technol China, Chengdu Womens & Childrens Cent Hosp, Sch Med, Dept Clin Lab, Chengdu, Peoples R China."

来源：BIOSENSORS & BIOELECTRONICS

ESI学科分类：CHEMISTRY

WOS号：WOS:000789664800002

JCR分区：Q1

影响因子：12.6

年份：2022

卷号：208

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：Post-PCR detection; Endonuclease; Strand displacement amplification; EGFR 19 exon deletion; CRISPR-Cas12a

摘要："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 endpoint 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."

基金机构："National Natural Science Foundation of China [81802115, 82172369, 81972025]; Basic Science and Cutting-edge Technology Research Projects of Chongqing Science and Technology Commission [cstc2019jcyj-msxmX0203, cstc2019jcyj-msxmX0848]; Sichuan Provincial Science and Technology Department Research Foundation of China [2020YFS0494]; Aesculap Academy Critical Care Scientific Research Fund [2016-12]"

基金资助正文："This research was supported by the National Natural Science Foundation of China (N.O. 81802115, 82172369, and 81972025), the Basic Science and Cutting-edge Technology Research Projects of Chongqing Science and Technology Commission (N.O. cstc2019jcyj-msxmX0203 and N.O. cstc2019jcyj-msxmX0848), the Sichuan Provincial Science and Technology Department Research Foundation of China (N.O. 2020YFS0494) and Aesculap Academy Critical Care Scientific Research Fund (2016-12)."