Motz M, Pääbo S, Kilger C
MPI for Evolutionary Anthropology, Leipzig, Germany.
Biotechniques. 2000 Aug;29(2):268-70. doi: 10.2144/00292st01.
A common problem in automated DNA sequencing when applying the Sanger chain termination method is ambiguous base calling caused by band compressions. Band compressions are caused by anomalies in the migration behavior of certain DNA fragments in the polyacrylamide gel because of intramolecular base pairing between guanine and cytosine residues. To reduce such undesired secondary structures, several modifications of the sequencing reaction parameters have been performed previously. Here, we have applied mixtures of the nucleotide analogs 7-deaza-dGTP and dITP instead of dGTP in the cycle sequencing reaction and in combination with varying buffer conditions. Band compressions were particularly well resolved, and reading length was optimal when a ratio of 7-deaza-dGTP:dITP of 4:1 was used in the in vitro DNA synthesis with AmpliTaq FS DNA polymerase. We conclude that the incorporation of both nucleotide analogs at these particular ratios leads to heterogeneous DNA chains that result in a reduction or elimination of intramolecular base pairing and thus a higher accuracy in the base assignment.
在应用桑格链终止法进行自动化DNA测序时，一个常见问题是由条带压缩导致的碱基误判。条带压缩是由于聚丙烯酰胺凝胶中某些DNA片段的迁移行为异常引起的，这是由于鸟嘌呤和胞嘧啶残基之间的分子内碱基配对所致。为了减少这种不期望的二级结构，之前已经对测序反应参数进行了几种修改。在这里，我们在循环测序反应中应用了核苷酸类似物7-脱氮-dGTP和dITP的混合物来代替dGTP，并结合不同的缓冲条件。当在使用AmpliTaq FS DNA聚合酶的体外DNA合成中使用7-脱氮-dGTP:dITP比例为4:1时，条带压缩得到了特别好的解决，并且读取长度是最佳的。我们得出结论，以这些特定比例掺入这两种核苷酸类似物会导致异质DNA链，从而减少或消除分子内碱基配对，进而提高碱基分配的准确性。
