Cloning of Xanthomonas campestris pv. campestris pathogenicity-related gene sequences by TAIL-PCR / 生物工程学报
Chinese Journal of Biotechnology
; (12): 182-186, 2002.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-231354
Biblioteca responsável:
WPRO
ABSTRACT
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.
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Assunto principal:
Virulência
/
DNA Bacteriano
/
Dados de Sequência Molecular
/
Sequência de Bases
/
Elementos de DNA Transponíveis
/
Reação em Cadeia da Polimerase
/
Mutagênese
/
Xanthomonas campestris
/
Clonagem Molecular
/
Proteínas de Fluorescência Verde
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2002
Tipo de documento:
Artigo