拟南芥的高通量定向诱导基因组局部突变技术

High-throughput TILLING for Arabidopsis.

作者信息

Till Bradley J, Colbert Trenton, Codomo Christine, Enns Linda, Johnson Jessica, Reynolds Steven H, Henikoff Jorja G, Greene Elizabeth A, Steine Michael N, Comai Luca, Henikoff Steven

机构信息

Basic Sciences Division, Fred Hutchison Cancer Research Center, Department of Biology, University of Washington, Seattle, USA.

出版信息

Methods Mol Biol. 2006;323:127-35. doi: 10.1385/1-59745-003-0:127.

DOI:10.1385/1-59745-003-0:127

PMID:16739573

Abstract

Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

摘要

基因组靶向诱导局部损伤技术（TILLING）是一种识别诱导点突变的通用策略，几乎可应用于任何生物体。在本章中，我们描述了高通量TILLING的基本方法。使用荧光标记引物扩增基因片段，产物变性并重新退火，以在突变序列与其野生型对应序列之间形成异源双链体。这些异源双链体是核酸内切酶CEL I切割的底物。切割后，使用LI-COR DNA分析仪系统在变性聚丙烯酰胺凝胶上分析产物。拟南芥TILLING项目（ATP）采用了高通量TILLING技术，为整个拟南芥研究群体提供点突变的等位基因系列。