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Journal of Clinical Microbiology, March 2001, p. 871-878, Vol. 39, No. 3
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.3.871-878.2001

Reverse Dot Blot Assay (Insertion Site Typing) for Precise Detection of Sites of IS6110 Insertion in the Mycobacterium tuberculosis Genome

Lauren M. Steinlein and Jack T. Crawford*

Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 14 September 2000/Returned for modification 29 November 2000/Accepted 31 December 2000

We have developed an amplification-based reverse dot blot assay for the detection of specific sites of insertion of the Mycobacterium tuberculosis insertion sequence IS6110. In this assay, a set of biotin-labeled amplicons representing the various copies of IS6110 and their flanking sequences is generated by linker-mediated PCR. The amplicons are then hybridized to immobilized oligonucleotide probes that are specific for known IS6110 insertion sites. The method was evaluated using an array of oligonucleotide probes corresponding to IS6110 insertion sites from M. tuberculosis strains CDC1551, Erdman, and H37Rv, and multidrug-resistant strain W. A set of 72 DNA samples from 60 M. tuberculosis clinical isolates was analyzed for the presence or absence of these insertion sites, and the assay was found to be highly reproducible. This method of identifying insertion sites has been named "insite" and can be used for the genotyping of M. tuberculosis complex strains based on IS6110 insertion site profiles.

* Corresponding author. Mailing address: Mailstop F08, NCID/DASTLR, CDC, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-1281. Fax: (404) 639-1287. E-mail: JCrawford{at}cdc.gov.

Journal of Clinical Microbiology, March 2001, p. 871-878, Vol. 39, No. 3
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.3.871-878.2001


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  • Kivi, M., Liu, X., Raychaudhuri, S., Altman, R. B., Small, P. M. (2002). Determining the Genomic Locations of Repetitive DNA Sequences with a Whole-Genome Microarray: IS6110 in Mycobacterium tuberculosis. J. Clin. Microbiol. 40: 2192-2198 [Abstract] [Full Text]  


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