[Cloning of Xanthomonas campestris pv. campestris pathogenicity-related gene sequences by TAIL-PCR].
Southern blot analysis with probe from mini-Tn5 gfp-km transposon indicated that 5 non-pathogenic mutants which were generated by insertion of mini-Tn5 gfp-km mutagenesis contained a single copy of the transposon. Using genomic DNA of each mutant as a template, TAIL-PCR was performed with seven arbitrary degenerate (AD) primers pairing with 3 nested specific primers designed based on the sequence of GFP toward outside in mini-Tn5 gfp-km. After 3-step PCR reactions, the flanking sequence of each mutant was obtained. The PCR product was ligated with pGEM-T EASY vector and then was transformed into E. coli DH5 alpha by electroporation. Positive clones were selected by white/blue colony and plasmid was isolated, then digested with EcoRI. Plasmid was sequenced if its insert was longer than 300 bp. Our results indicated that TAIL-PCR was proved to be a simple and efficient approach in identification of gene using insertion mutagenesis.
Ying
Ge
G
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Wu
Wei
W
He
Chao-Zu
CZ
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000302:isolation & purification; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods; D016959:Xanthomonas campestris / Q000235:genetics* / Q000472:pathogenicity
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins