High-throughput method for detecting genomic-deletion polymorphisms.

TitleHigh-throughput method for detecting genomic-deletion polymorphisms.
Publication TypeJournal Article
Year of Publication2004
Authorsde la Salmonière Y-OLuc Goguet, Kim CC, Tsolaki AG, Pym AS, Siegrist MS, Small PM
JournalJ Clin Microbiol
Date Published2004 Jul
KeywordsDNA Probes, Genome, Bacterial, Mycobacterium tuberculosis, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Polymorphism, Genetic

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.

Alternate JournalJ. Clin. Microbiol.
PubMed ID15243038
PubMed Central IDPMC446289
Grant ListAI34238 / AI / NIAID NIH HHS / United States