Sequential amplification of flanking sequences by Y-shaped adaptor dependent extension using multiple templates.
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.
Xiao
Yue-Hua
YH
Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing 400716, China.
Peng
Yi
Y
Luo
Ming
M
Li
De-Mou
DM
Hou
Lei
L
Pei
Yan
Y
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
D018744:DNA, Plant; D004262:DNA Restriction Enzymes