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Rapid Polymerase Chain Reaction Screening of Helicobacter pylori Chromosomal Point Mutations
Author(s) -
Ge Zhongming,
Taylor Diane E.
Publication year - 1997
Publication title -
helicobacter
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.206
H-Index - 79
eISSN - 1523-5378
pISSN - 1083-4389
DOI - 10.1111/j.1523-5378.1997.tb00073.x
Subject(s) - polymerase chain reaction , point mutation , polymerase chain reaction optimization , polymerase , nested polymerase chain reaction , genetics , microbiology and biotechnology , chemistry , mutation , biology , dna , gene
Background. Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vac A and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains. Materials and Methods. The cop P gene from H. pylori UA802 was used as a model for developing a mutation screening method. Four point mutations were introduced into the cop P gene by in vitro site‐directed mutagenesis and were verified by DNA sequencing. The mutated cop P gene replaced the wild‐type locus by natural transformation and homologous recombination. The site‐specific mutants were screened by polymerase chain reaction (PCR) using 3′‐end mismatched primers. The origins of the PCR fragments were demonstrated by Southern hybridization with the cop P‐derived DNA probe. Results. Three of these four mutations were characterized by PCR with the specific primers that contained the 3′‐terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates. One mutation was able to be identified with the foregoing primer containing an additional wild‐type nucleotide at its 3′‐end. Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions. To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted. Conclusion. The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site‐specific mutations on both plasmids and chromosomal DNA. Although the method was developed by using a specified H. pylori gene, it can be extended easily to other genes of interest in H. pylori or other organisms.