Burland V, Daniels D L, Plunkett G, Blattner F R
Laboratory of Genetics, University of Wisconsin, Madison 53706.
Nucleic Acids Res. 1993 Jul 25;21(15):3385-90. doi: 10.1093/nar/21.15.3385.
The design of large scale DNA sequencing projects such as genome analysis demands a new approach to sequencing strategy, since neither a purely random nor a purely directed method is satisfactory. We have developed a strategy that combines these two methods in a way that preserves the advantages of both while avoiding their particular limitations. Computer simulations showed that a specific balance of random and directed sequencing was required for the most efficient strategy, termed the Janus strategy, which has been used in the Escherichia coli genome sequencing project. This approach depended on obtaining sequence easily from either strand of a cloned insert, and was facilitated by inversion of the insert in the engineered M13 vector Janus, by site-specific recombination. The inversion was accomplished simply by growth on the appropriate host strain, when the DNA strand incorporated into the new single stranded phage was complementary to that in the original phage, and was sequenced by the same simple protocol as the first strand.
诸如基因组分析之类的大规模DNA测序项目的设计需要一种新的测序策略方法，因为单纯的随机方法或单纯的定向方法都不尽人意。我们开发了一种策略，将这两种方法结合起来，既保留了两者的优点，又避免了它们各自的局限性。计算机模拟表明，对于最有效的策略（称为雅努斯策略），需要随机测序和定向测序的特定平衡，该策略已用于大肠杆菌基因组测序项目。这种方法依赖于从克隆插入片段的任何一条链轻松获得序列，并且通过在工程化的M13载体雅努斯中通过位点特异性重组使插入片段反向而得以实现。当掺入新单链噬菌体的DNA链与原始噬菌体中的DNA链互补时，只需在适当的宿主菌株上生长即可完成反向操作，并通过与第一条链相同的简单方案进行测序。
