Wang Dafang, Peterson Thomas
Iowa State University, Ames, IA, USA.
Methods Mol Biol. 2013;1057:117-22. doi: 10.1007/978-1-62703-568-2_8.
Localizing Ac insertions is a fundamental task in studying Ac-induced mutation and chromosomal rearrangements involving Ac elements. Researchers may sometimes be faced with the situation in which the sequence flanking one side of an Ac/Ds element is known, but the other flank is unknown. Or, a researcher may have a small sequence surrounding the Ac/Ds insertion site and needs to obtain additional flanking genomic sequences. One way to rapidly clone unknown Ac/Ds flanking sequences is via a PCR-based method termed Ac casting. This approach utilizes the somatic transposition activity of Ac during plant development, and provides an efficient means for short-range genome walking. Here we describe the principle of Ac casting, and show how it can be applied to isolate Ac macrotransposon insertion sites.
定位Ac插入是研究Ac诱导的突变以及涉及Ac元件的染色体重排的一项基本任务。研究人员有时可能会面临这样的情况：已知Ac/Ds元件一侧的侧翼序列，但另一侧未知。或者，研究人员可能有围绕Ac/Ds插入位点的一小段序列，需要获取额外的侧翼基因组序列。快速克隆未知Ac/Ds侧翼序列的一种方法是通过一种基于PCR的方法，称为Ac铸型。这种方法利用了植物发育过程中Ac的体细胞转座活性，并为短距离基因组步移提供了一种有效的手段。在这里，我们描述了Ac铸型的原理，并展示了它如何应用于分离Ac大转座子插入位点。
