title: Removing PCR for the elimination of undesired DNA fragments cycle by cycle.
abstract: 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 - ApeKI, 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, ApeKI 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.
authors.affiliation: Department of Plant Pathology, College of Plant Science and Technology and the Key Lab of Crop Disease Monitoring & Safety Control in Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China.
authors.forename: Jiaojiao
authors.initials: J
authors.lastname: Huan
authors.forename: Kangkang
authors.initials: K
authors.lastname: Wan
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Liu
authors.forename: Wubei
authors.initials: W
authors.lastname: Dong
authors.forename: Guoying
authors.initials: G
authors.lastname: Wang
mesh_terms: D059018:DNA Contamination; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D004262:DNA Restriction Enzymes
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