生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (2): 103-.doi: 10.3969/j.issn.2095-1736.2024.02.103

• 技术方法 • 上一篇    下一篇

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

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0.003~0.006 μmol/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。   

  1. 东华大学 生物医学工程学院， 上海 201620
  • 出版日期:2024-04-18 发布日期:2024-04-17
  • 通讯作者: 肖君华，博士，教授，研究方向为分子遗传学，E-mail:xiaojunhua@dhu.edu.cn
  • 作者简介:沈泽嘉，硕士研究生，研究方向为分子检测，E-mail:stu_szj@163.com
  • 基金资助:
    国家自然科学基金项目（31772550）； 上海市科委基金资助项目（17140903102）

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

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 0.003-0.006 μmol/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.   

  1. School of Biomedical Engineering, Donghua University, Shanghai 201620, China
  • Online:2024-04-18 Published:2024-04-17

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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0.003~0.006 μmol/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。

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

Abstract: 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 0.003-0.006 μmol/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.

Key words: allele-specific PCR, multiplex PCR, high-fidelity Taq enzyme, genotype, chromosome substitution mice

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