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.
作者
李书亚
郜艳敏
胡亚赛
郝敏
齐浩
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)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
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
