Mutagenesis during in vitro DNA synthesis.
Mutagenesis during in vitro DNA synthesis.
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体外 DNA 合成过程中的诱变。
DOI：
10.1073/pnas.75.4.1924
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发表时间：
1978
影响因子：
11.1
通讯作者：
L. Loeb
中科院分区：
文献类型：
--
作者：
L. Weymouth;L. Loeb
使用天然 DNA 模板进行体外 DNA 合成的错误频率已通过核苷酸取代的生物测定进行了测量。含有琥珀突变的phiX174 DNA在体外由大肠杆菌DNA聚合酶I复制，并通过转染大肠杆菌原生质球测定子代DNA的回复频率。在标准反应条件下，大肠杆菌聚合酶 I 在 am3 基因座上每掺入 7700 个核苷酸，错误少于 1 个。用 Mn2+ 替代 Mg2+ 以及不等浓度的脱氧核苷三磷酸底物可将突变频率提高到千分之一以上。因此，大肠杆菌 DNA 聚合酶 I 可以高保真度复制天然 DNA 模板，其准确性可能会受到反应条件改变的影响。
The error frequency of in vitro DNA synthesis using a natural DNA template has been measured with a biological assay for nucleotide substitutions. phiX174 DNA containing an amber mutation was copied in vitro by Escherichia coli DNA polymerase I, and the reversion frequency of the progeny DNA was determined by transfection of E. coli spheroplasts. E. coli polymerase I makes less than 1 mistake at the am3 locus for every 7700 nucleotides incorporated under standard reaction conditions. Substitution of Mn2+ for Mg2+ and unequal concentrations of deoxynucleoside triphosphate substrates raises this mutation frequency to greater than 1 in 1000. Thus, E. coli DNA polymerase I can copy natural DNA templates with high fidelity and its accuracy can be affected by alterations in reaction conditions.