Xiao Yue-Hua, Peng Yi, Luo Ming, Li De-Mou, Hou Lei, Pei Yan
Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing 400716, China.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2007 Feb;33(1):85-90.
Y-shaped adaptor dependent extension (YADE) method is a useful tool to amplify the flanking sequence of a known DNA sequence, but its efficiency is frequently limited by the restriction sites around the known sequence. In this paper, we demonstrated that using multiple templates derived from several restrictions and ligations could dramatically increase the efficiency of YADE method and render it suitable for sequential amplification of flanking sequences. With templates originating from 7 digestions, a 2,228-bp 5'-upstream sequence of a cotton small GTPase gene was obtained by two rounds of sequential YADE amplifications. The results demonstrated that the YADE method with multiple templates may be a useful tool for sequential PCR walking in complex genomes.
Y形接头依赖延伸（YADE）方法是扩增已知DNA序列侧翼序列的有用工具，但其效率常常受到已知序列周围限制酶切位点的限制。在本文中，我们证明使用源自多次酶切和连接的多个模板可显著提高YADE方法的效率，并使其适用于侧翼序列的连续扩增。通过两轮连续的YADE扩增，利用源自7次酶切的模板获得了棉花小GTP酶基因2228 bp的5'上游序列。结果表明，具有多个模板的YADE方法可能是在复杂基因组中进行连续PCR步移的有用工具。
