Mutation Analysis of L-Thymidine-Induced Replication Products Using a Restriction Enzyme–Mediated Assay

Q4 Chemistry Current Protocols in Nucleic Acid Chemistry Pub Date : 2020-12-08 DOI:10.1002/cpnc.121

Yuhe Kan, Li Wu, Yujian He

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引用次数: 1

Abstract

This article describes experimental and analytical procedures for evaluating the efficiency and fidelity of DNA replication containing mirror-image thymidine (L-T) in E. coli. The procedure involves construction of DNA recombinants containing a restriction enzyme (PstI) recognition site in which the L-T lesion is site-specifically located within the PstI recognition sequence (CTGCAG). The recombinants are transfected into DH5α cells. DNA is extracted, amplified, and cleaved into relatively short fragments using different combinations of restriction enzymes to facilitate electrophoretic analysis. Detailed explanations for the restriction enzyme–mediated assay for detection of mutagenic properties of mirror-image thymidine at a predetermined site are also presented. Advantages and limitations of the assay are discussed by comparing it to other techniques used for detecting lesion-induced mutation efficiency, and a troubleshooting guide is provided. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of oligonucleotides containing L-T

Basic Protocol 2: Construction of DNA recombinants

Basic Protocol 3: Mutation analysis of L-T-induced replication products using a restriction enzyme–mediated assay

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利用限制性内切酶介导的实验分析l -胸腺嘧啶诱导的复制产物的突变

本文描述了在大肠杆菌中评估含有镜像胸苷(L-T)的DNA复制效率和保真度的实验和分析方法。该过程包括构建含有限制性内切酶(PstI)识别位点的DNA重组体，其中L-T病变位点特异性位于PstI识别序列(CTGCAG)内。将重组体转染DH5α细胞。DNA被提取，扩增，并使用不同的限制性内切酶组合切割成相对较短的片段，以促进电泳分析。详细解释了限制性内切酶介导的检测镜像胸苷在预定位点的诱变特性的测定。通过将其与用于检测病变诱导突变效率的其他技术进行比较，讨论了该测定的优点和局限性，并提供了故障排除指南。©2020 Wiley期刊公司基本方案1:含l - t寡核苷酸的合成基本方案2:DNA重组的构建基本方案3:使用限制性内切酶介导的测定法对l - t诱导的复制产物进行突变分析

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Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry

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期刊介绍： Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.

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