Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.
Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.
复制标题
通过酶不匹配的裂解从合成基因中去除不匹配的碱基。
DOI：
10.1093/nar/gni058
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发表时间：
2005-03-30
影响因子：
14.9
通讯作者：
Hegemann, Peter
中科院分区：
文献类型：
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作者：
Fuhrmann, Markus;Oertel, Wolfgang;Berthold, Peter;Hegemann, Peter
长多核苷酸从头合成的成功在很大程度上取决于所用寡核苷酸的质量和纯度。通常，任何合成反应的初级产物都是直接克隆的，并且必须鉴定具有正确产物的克隆。在这项研究中，已经建立了一种新的策略，用于从初级基因合成产物中去除不需要的序列变体。用特异性核酸内切酶切割单碱基对错配、插入和缺失。三种不同的酶-T7核酸内切酶I，T4核酸内切酶VII和大肠杆菌核酸内切酶V-已被测试。以氯霉素乙酰转移酶（cat）为模型，采用不同的寡核苷酸连接法一步合成多核苷酸，合成了编码cat的多核苷酸。通过合成cat的功能性克隆并将错误率与未处理产物的错误率进行比较，分析了酶错配切割（EMC）作为错误校正步骤对正确产物频率的影响。观察到所有突变类型的显著减少。统计分析显示，T4和大肠杆菌核酸内切酶减少了克隆的合成基因产物中突变的发生。EMC处理是成功的，特别是在从初级连接产物中去除缺失和插入方面。
The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes—T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V—have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.