Shao Hongguang, Lok James B
Department of Pathobiology, University of Pennsylvania, Philadelphia, USA.
Bio Protoc. 2014 Jan 5;4(1). doi: 10.21769/BioProtoc.1015.
Splinkerette PCR (spPCR) is a newly developed and efficient method to ascertain and characterize genomic insertion sites of transgenes. The method described in this protocol was successfully applied to confirm transposon-mediated integration of transgenes into chromosomes of the parasitic nematode . This work is described in detail in Shao . (2012) and presented here in a simplified diagram (Figure 1). Using this method, chromosomal loci of integration were determined based on target site and 5'- and 3' flanking sequences. Therefore, spPCR can be a useful method to confirm integrative transgenesis in functional genomic studies of parasitic nematodes. Potter and Luo (2010) contains a protocol for use of spPCR to detect and map transposon-mediated chromosomal integrations in , and was the source of our method for . The splinkerette- and specific oligos described in that reference could be used without modification in . For interested readers, a general review of the biology of parasitic nematodes in the genus may be found in Viney and Lok (2007), and a methods-based article on as an experimental model, with information on transgenesis, may be found in Lok (2007).
拼接式PCR（spPCR）是一种新开发的高效方法，用于确定转基因的基因组插入位点并对其进行表征。本方案中描述的方法已成功应用于确认转座子介导的转基因整合到寄生线虫的染色体中。邵（2012年）对这项工作进行了详细描述，并在此以简化图（图1）呈现。使用该方法，基于靶位点以及5'和3'侧翼序列确定整合的染色体位点。因此，在寄生线虫的功能基因组学研究中，spPCR可能是一种用于确认整合转基因的有用方法。波特和罗（2010年）包含了使用spPCR检测和定位转座子介导的染色体整合的方案，并且是我们用于[此处缺失具体内容]的方法的来源。该参考文献中描述的拼接式引物和特异性寡核苷酸可在[此处缺失具体内容]中直接使用，无需修改。对于感兴趣的读者，可在维内和洛克（2007年）中找到关于[此处缺失具体线虫属名称]寄生线虫生物学的综述，在洛克（2007年）中可找到一篇以[此处缺失具体线虫名称]作为实验模型的基于方法的文章，其中包含转基因相关信息。
