构建大插入片段的酵母人工染色体文库。

Construction of YAC libraries with large inserts.

作者信息

Foote Simon, Denny Christopher

机构信息

Whitehead Institute, Cambridge, Massachusetts, USA.

出版信息

Curr Protoc Hum Genet. 2002 Feb;Chapter 5:Unit5.2. doi: 10.1002/0471142905.hg0502s31.

DOI:10.1002/0471142905.hg0502s31

PMID:18428331

Abstract

The yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase-pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease-EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.

摘要

酵母人工染色体（YAC）克隆系统使将大片段基因组DNA克隆到酵母中成为可能。已经构建了包含插入片段在兆碱基对范围内的克隆的文库。本单元中的基本方案描述了YAC载体的制备以及将连接的DNA转化到酵母原生质球中的方法。一个支持方案描述了裂解酶滴定以制备原生质球。其他支持方案详细介绍了基因组DNA部分消化的两种方法：分别是EcoRI限制性内切酶-EcoRI甲基化酶竞争法和使用限量Mg2+对基因组DNA进行部分消化的方法。