PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Natalia S. Akopyants, Arkady Fradkov, Luda Diatchenko, Jason E. Hill, Paul D. Siebert, Sergey A. Lukyanov, Eugene D. Sverdlov, Douglas E. Berg

Research output: Contribution to journalArticle

192 Scopus citations

Abstract

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had hornology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Original languageEnglish
Pages (from-to)13108-13113
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number22
DOIs
StatePublished - Oct 27 1998

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