Till Bradley J, Colbert Trenton, Tompa Rachel, Enns Linda C, Codomo Christine A, Johnson Jessica E, Reynolds Steven H, Henikoff Jorja G, Greene Elizabeth A, Steine Michael N, Comai Luca, Henikoff Steven
BasicSciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Methods Mol Biol. 2003;236:205-20. doi: 10.1385/1-59259-413-1:205.
Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, 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.
定向诱导基因组局部突变（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。在此，我们描述高通量TILLING的基本方法。使用荧光标记引物扩增基因片段，产物经变性和复性形成突变序列与其野生型对应序列之间的异源双链体。这些异源双链体是核酸内切酶CEL I的切割底物。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，为整个拟南芥研究群体提供点突变的等位基因系列。
