Siemieniak D R, Sieu L C, Slightom J L
Molecular Biology Research-Unit 7242, Upjohn Company, Kalamazoo, Michigan 49007.
Anal Biochem. 1991 Feb 1;192(2):441-8. doi: 10.1016/0003-2697(91)90562-8.
The primer-directed enzymatic sequencing method for sequencing double-stranded DNA templates has made possible the development of new strategies for directly sequencing large DNA molecules. Toward this goal, we have developed a strategy and the necessary techniques to obtain the complete sequence of cosmid clones (double-stranded DNA molecules in the size range of 50 kb). Our present strategy uses the chemical sequencing method to obtain sequence initiation points internal to a cosmid insert and the primer-directed enzymatic DNA sequencing method to extend these sequence contigs. As part of this development we added a nucleotide "chase" solution to the standard T7 sequencing protocol and included the use of both [alpha-32P]-dATP and -dCTP for labeling. With these modifications our double-stranded cosmid DNA sequencing reactions routinely extend well beyond 1000 bp, and film exposure times are kept to a minimum (24 to 48 h). We can routinely separate sequenced DNA fragments, using a 1-m gel system, which can be accurately read (with less than 0.5% error) to distances of 800 bp or more, from the oligomer primer. The strategy and procedures presented here allow the complete sequence of a cosmid clone to be obtained without subcloning.
用于双链DNA模板测序的引物引导酶促测序方法，使得直接对大DNA分子进行测序的新策略得以发展。为实现这一目标，我们开发了一种策略及必要技术，以获得黏粒克隆（大小在50 kb范围内的双链DNA分子）的完整序列。我们目前的策略是使用化学测序方法获得黏粒插入片段内部的序列起始点，并使用引物引导酶促DNA测序方法来延伸这些序列重叠群。作为此项开发工作的一部分，我们在标准的T7测序方案中加入了核苷酸“追踪”溶液，并同时使用[α-32P]-dATP和-dCTP进行标记。通过这些改进，我们的双链黏粒DNA测序反应通常能延伸超过1000 bp，且胶片曝光时间可保持在最短（24至48小时）。我们可以常规地使用1米长的凝胶系统分离已测序的DNA片段，该系统能够从寡聚引物准确读取（误差小于0.5%）至800 bp或更长距离。本文介绍的策略和程序使得无需亚克隆即可获得黏粒克隆的完整序列。
