title: Sequential amplification of flanking sequences by Y-shaped adaptor dependent extension using multiple templates.
abstract: 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.
authors.affiliation: Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing 400716, China.
authors.forename: Yue-Hua
authors.initials: YH
authors.lastname: Xiao
authors.forename: Yi
authors.initials: Y
authors.lastname: Peng
authors.forename: Ming
authors.initials: M
authors.lastname: Luo
authors.forename: De-Mou
authors.initials: DM
authors.lastname: Li
authors.forename: Lei
authors.initials: L
authors.lastname: Hou
authors.forename: Yan
authors.initials: Y
authors.lastname: Pei
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018744:DNA, Plant / Q000032:analysis*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic
chemical_list: D018744:DNA, Plant; D004262:DNA Restriction Enzymes