额外的错配碱基提高T4 DNA连接酶等位特异性连接（英文）  

Systematic Evaluation of the Improvement of Allele-Specific Ligation by T4 DNA Ligase by an Additional Mismatched Base Pair

作　　者：李书亚 郜艳敏 胡亚赛 郝敏 齐浩[1,2] LI Shu-Ya;GAO Yan-Min;HU Ya-Sai;HAO Min;QI Hao(School of Chemical Engineering and Technology,Tianjin Universit;Key Laboratory of Systems Bioengineering（Ministry of Education）,Tianfin University,Tianfia 300072,China)

机构地区：[1]天津大学化工学院 [2]系统生物工程教育部重点实验室,天津大学,天津300072

出　　处：《中国生物化学与分子生物学报》2018年第8期854-860,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.21476167和No.21778039)资助

摘　　要：连接是一种主要的DNA处理过程。由于较低的商业成本以及核酸底物识别的灵活性,T4DNA连接酶被广泛应用于生物分子工程,特别是特定核酸序列的等位特异性连接检测。本文评估了在T4 DNA连接酶介导的连接反应中,引入额外的错配碱基对所产生的影响。设计了超过150组DNA/DNA或DNA/RNA带有的额外错配碱基对的组合。结果发现,引入额外的错配碱基对后,T4 DNA连接酶在DNA/DNA连接中特异性可提高60倍以上,而在DNA/RNA连接中特异性只能提高2倍。在等位特异性连接中,有的错配碱基对可使T4 DNA连接酶的特异性提高600多倍。Ligation is a fundamental DNA-modification process. Due to the low commercial cost and flexibility in nucleotide substrate recognition, T4 DNA ligase is widely utilized in biomolecular engineering applications,especially in allele-specific ligation for the detection of specific nucleotide sequences. In this study,we evaluated the influence of an introduced additional mismatched base pair on the specificity of T4 DNA ligase-mediated DNA ligation. More than 150 combinations of hybridized DNA/DNA or DNA/RNA structures with designed mismatched base pairs were systematically quantified,demonstrating that an additional mismatch significantly improved the specificity of T4 DNA ligase more than 60-fold in hybridized DNA/DNA structures compared with 2-fold in DNA/RNA structures. The effect on DNA pairs was much greater than that in RNA targets. The optimized mismatched base pair was able to improve the specificity of T4 DNA ligase-mediated allele-specific ligation over 600-fold.

关 键 词：等位特异性连接 错配 T4 DNA连接酶 锁式探针 

分 类 号：Q7[生物学—分子生物学]