基于新型高保真Taq酶的多重等位基因特异性PCR方法的建立与应用  

Establishment and application of a multiple allele-specific PCR method based on a novel high-fidelity Taq polymerase

作　　者：沈泽嘉 徐逸梦 时景景 周宇荀[1] 李凯[1] 肖君华[1] SHEN Zejia;XU Yimeng;SHI Jingjing;ZHOU Yuxun;LI Kai;XIAO Junhua(School of Biomedical Engineering,Donghua University,Shanghai 201620,China)

机构地区：[1]东华大学生物医学工程学院,上海201620

出　　处：《生物学杂志》2024年第2期103-109,115,共8页Journal of Biology

基　　金：国家自然科学基金项目(31772550);上海市科委基金资助项目(17140903102)。

摘　　要：在8号染色体存在部分替换片段的小鼠上挑选3个SNP位点进行初步方案检验,包括高保真Taq酶的特异性测试以及引物浓度对分型结果的影响,并评价该方法的灵敏度;之后应用该技术检测野生1号染色体替换系小鼠上的9个SNP的不同样本。对Taq酶的特异性研究表明:引物在未引入突变的前提下检测结果与测序结果保持一致;引物稀释实验说明浓度最低为0.003~0.006μmol/L,灵敏度的检测结果表示最低浓度检测限可达0.07 ng/μL以下。最后检测得到5种标准样本的基因型,其代表9个SNP的组合。成功验证并建立该新型高特异性Taq酶应用于多重等位基因特异性PCR实验的总体流程。提供一套具备高特异性、能够针对多个SNP位点进行检测的低成本方案,对多重等位基因特异性PCR技术的开发同样具有参考价值。This technology was established on the basis of three selected SNP loci for preliminary testing on mice with partially replaced fragments on chromosome 8,including specificity testing of high-fidelity Taq enzymes and evaluation of the effect of primer concentration on genotyping results as well as sensitivity assessment.It was used to detect nine SNPs in different wild-type chromosome 1 substitution mouse samples.The study showed that the detection results without introducing mutations into primers were consistent with sequencing results.The dilution experiments indicated that the optimal concentration was at least 0.003-0.006μmol/L,while sensitivity detection revealed that the minimal concentration detection limit could reach below 0.07 ng/μL.Finally,five standard samples’genotypes representing combinations of nine SNPs were detected.The overall process for applying this highly specific Taq enzyme in multiple allele-specific PCR experiments was eventually validated and established.This article offered a low-cost solution with high specificity and the ability to detect multiple SNP loci,which preserved the reference value for the development of multiple allele-specific PCR technology.

关 键 词：等位基因特异性PCR 多重PCR 高保真Taq酶 基因分型 染色体替换系小鼠 

分 类 号：Q75[生物学—分子生物学]