Segments missing from the draft human genome sequence can be isolated by transformation-associated recombination cloning in yeast
Segments missing from the draft human genome sequence can be isolated by transformation-associated recombination cloning in yeast
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DOI：
10.1038/sj.embor.embor766
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发表时间：
2003-03-01
期刊：
影响因子：
7.7
通讯作者：
Larionov, V
中科院分区：
文献类型：
--
作者：
Kouprina, N;Leem, SH;Larionov, V
报告的人类基因组序列草图包括许多由未知序列的间隙分隔的重叠群。这些缺口可能是由于用于 DNA 测序的大肠杆菌文库中不存在的染色体区域造成的，因为它们无法在细菌中有效克隆（如果有的话）。使用在酵母中生成的酵母人工染色体 (YAC)/细菌人工染色体 (BAC) 文库，我们发现测试的约 6% 的人类 DNA 序列转化大肠杆菌细胞的效率低于酵母细胞，并且在大肠杆菌中的稳定性低于酵母。当对酵母中克隆的几个 YAC/BAC 分离株的末端进行测序并与报道的草图序列进行比较时，发现那些低效转化大肠杆菌细胞的 YAC/BAC 分离株的序列存在重大不一致。通过转化相关重组 (TAR) 克隆从人类 DNA 中重新分离出两个人类基因组片段。对这些区域的重新测序表明，草稿中的错误是在大肠杆菌中克隆过程中错误组装和特定 DNA 序列丢失的结果。这些结果表明，TAR 克隆可能是一种有价值的方法，可以在人类基因组计划的最后阶段广泛使用。
The reported draft human genome sequence includes many contigs that are separated by gaps of unknown sequence. These gaps may be due to chromosomal regions that are not present in the Escherichia coli libraries used for DNA sequencing because they cannot be cloned efficiently, if at all, in bacteria. Using a yeast artificial chromosome (YAC)/ bacterial artificial chromosome (BAC) library generated in yeast, we found that approximately 6% of human DNA sequences tested transformed E. coli cells less efficiently than yeast cells, and were less stable in E. coli than in yeast. When the ends of several YAC/BAC isolates cloned in yeast were sequenced and compared with the reported draft sequence, major inconsistencies were found with the sequences of those YAC/BAC isolates that transformed E. coli cells inefficiently. Two human genomic fragments were re-isolated from human DNA by transformation-associated recombination (TAR) cloning. Re-sequencing of these regions showed that the errors in the draft are the results of both missassembly and loss of specific DNA sequences during cloning in E coli. These results show that TAR cloning might be a valuable method that could be widely used during the final stages of the Human Genome Project.