Error analysis of chemically synthesized polynucleotides
Error analysis of chemically synthesized polynucleotides
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DOI：
10.2144/98242st01
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发表时间：
1998-02-01
期刊：
影响因子：
2.7
通讯作者：
Rill, RL
中科院分区：
文献类型：
--
作者：
Hecker, KH;Rill, RL
两个单链多核苷酸构建体。长度为123和126个核苷酸，使用标准亚磷酰胺化学方法化学合成。通过用DNA聚合酶进行引物延伸并用两种限制性内切酶进行末端修剪，从多核苷酸制备约100-bp长的可克隆的双链DNA片段，然后将片段连接到单独的质粒中。在质粒的体内扩增后，通过双脱氧测序确定单个插入拷贝中的错误。测序的10个插入片段中有5个含有错误，包括7个单碱基对缺失，1个四碱基对缺失和1个G->C颠换。后两种错误的起源尚不清楚，但单碱基缺失与聚合酶的错误不一致;因此，化学合成中最常见的序列错误是缺失突变。缺失最有可能是由不完全加帽或脱三苯甲基化引起的。在依赖于单个插入克隆中多核苷酸序列的正确性的应用中，观察到的错误率可能成为显著的限制因素。
Two single-stranded polynucleotide constructs. 123 and 126 nucleotides in length, were chemically synthesized using standard phosphoramidite chemistry. Clonable, double-stranded DNA fragments about 100-bp long were prepared from the polynucleotides by primer extension with a DNA polymerase and end-trimming with two restriction endonucleases, then the fragments were ligated into separate plasmids. Errors in individual insert copies were determined by dideoxy sequencing after in vivo amplification of plasmids. Five of the ten inserts sequenced contained errors, including seven single-base-pair deletions, one four-base-pair deletion and one G-->C transversion. The origins of the latter two errors are unclear, but single-base deletions are inconsistent with errors of polymerases; thus, the most common sequence errors of chemical synthesis are deletion mutations. Deletions are most likely to result from incomplete capping or de-tritylation. The observed error rate can become a significant limiting factor in applications that depend on the correctness of a polynucleotide sequence in individual insert clones.