State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
Biotechniques. 2011 Dec;51(6):421-3. doi: 10.2144/000113787.
SiteFinding-PCR is a method for isolating flanking sequence tags (FSTs) of T-DNA insertion lines, but the efficiency needs to be improved. Here we report a computation-assisted design for the random primers used in SiteFinding- PCR. A short sequence, GCATG, was screened from the rice genome and used as the 3' end of the random primer. When applying the optimized primer for isolating FSTs from 168 transgenic rice lines, we obtained 107 specific products, including 64 FSTs. The efficiency of obtaining FSTs using the modified version of SiteFinding-PCR increased by 73.0% compared with the method previously reported (P < 0.01, µ test). We also provide computational results for several other plant species such as maize, sorghum, Arabidopsis, foxtail millet, and Brachypodium based on the available genome data, so that the modified method could be easily adapted to other species.
位点发现-PCR 是一种分离 T-DNA 插入系侧翼序列标签（FST）的方法，但需要提高其效率。在这里，我们报告了一种用于位点发现-PCR 随机引物的计算辅助设计。从水稻基因组中筛选出短序列 GCATG，并将其用作随机引物的 3'端。当应用优化的引物从 168 个转基因水稻系中分离 FST 时，我们获得了 107 个特异性产物，包括 64 个 FST。与之前报道的方法相比（P < 0.01，µ 检验），使用改良的位点发现-PCR 获得 FST 的效率提高了 73.0%。我们还根据现有基因组数据为玉米、高粱、拟南芥、谷子和短柄草等其他几种植物提供了计算结果，以便将改良的方法轻松应用于其他物种。
