Ahmed A, Podemski L
Department of Biological Sciences, University of Alberta, Edmonton, Canada.
Gene. 1997 Sep 15;197(1-2):367-73. doi: 10.1016/s0378-1119(97)00285-0.
Previous attempts to use the non-random approach for sequencing long DNA fragments have met with little success. As a result, nearly all genomic sequencing is done by the random (shotgun) approach, and the economy promised by the non-random approach has so far not materialized. Here we describe a simple system based on the use of ordered deletions that can be incorporated in the common strategies for genome sequencing. Long genomic fragments are cloned in the pAL-F cosmid and fragmented by digestion with specific restriction endonucleases. The digests are religated to subclone individual restriction fragments. The subclones are then subdivided by overlapping deletions and used for sequencing. We present the nucleotide sequences of two cosmid inserts from chromosome IV of Drosophila (containing the ci gene and the 5' end of the zfh-2 gene) that were determined by this method. This is the first report of successful sequencing of long genomic fragments by the use of overlapping deletions. Our calculations show that, with the present approach, sequence data can be acquired at a rate comparable to the shotgun approach but with significantly reduced numbers (approximately 30%) of sequencing runs. Hence, the use of ordered deletions should allow significant savings in both the amount and cost of sequencing work.
以往尝试使用非随机方法对长DNA片段进行测序，但收效甚微。因此，几乎所有的基因组测序都是通过随机（鸟枪法）方法完成的，而非随机方法所承诺的经济性至今尚未实现。在此，我们描述了一个基于有序缺失的简单系统，该系统可纳入基因组测序的常用策略中。长基因组片段被克隆到pAL-F粘粒中，并用特定的限制性内切酶消化使其片段化。消化产物重新连接以亚克隆各个限制性片段。然后通过重叠缺失对亚克隆进行细分并用于测序。我们展示了通过这种方法确定的来自果蝇第四条染色体的两个粘粒插入片段的核苷酸序列（包含ci基因和zfh-2基因的5'端）。这是关于使用重叠缺失成功对长基因组片段进行测序的首次报道。我们的计算表明，采用目前的方法，可以以与鸟枪法相当的速率获取序列数据，但测序运行次数显著减少（约30%）。因此，使用有序缺失应该能够在测序工作的数量和成本方面实现大幅节省。
