文章基本信息

标题：Removing PCR for the elimination of undesired DNA fragments cycle by cycle
本地全文：下载
作者：Jiaojiao Huan ; Kangkang Wan ; Yunjun Liu 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2013
卷号：3
期号：1
DOI：10.1038/srep02303
语种：English
出版社：Springer Nature
摘要：A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ~ 100% (four DNA fragments were tested). Major components of the R-PCR include drivers, a thermostable restriction enzyme - ApeK I, and a poly(dA) adapter with mismatched restriction enzyme recognition sites. Drivers were generated from the undesired genes. In each cycle of R-PCR, drivers anneal to complementary sequences and allow extension by Taq DNA polymerase. Thus, ApeK I restriction sites in the undesired genes are recovered, and adapters of these undesired DNA fragments are removed. Using R-PCR, we isolated maize upregulated defense-responsive genes and Blumeria graminis specialized genes, including key pathogenesis-related effectors. Our results show that after the R-PCR reaction, most undesired genes, including very abundant genes, became undetectable. The R-PCR is an easy and cost-efficient method to eliminate undesired genes and clone desired genes.