Cloning of Xanthomonas campestris pv. campestris pathogenicity-related gene sequences by TAIL-PCR.
- Author:
Ge YING
1
;
Wei WU
;
Chao-Zu HE
Author Information
1. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Cloning, Molecular;
methods;
DNA Transposable Elements;
DNA, Bacterial;
isolation & purification;
Genes, Bacterial;
Green Fluorescent Proteins;
Luminescent Proteins;
genetics;
Molecular Sequence Data;
Mutagenesis;
Polymerase Chain Reaction;
methods;
Xanthomonas campestris;
genetics;
pathogenicity
- From:
Chinese Journal of Biotechnology
2002;18(2):182-186
- CountryChina
- Language:Chinese
-
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.
