Protocol

Mycobacteria Protocols

Volume 465 of the series Methods in Molecular Biology pp 353-370

Date:

Insertion Element IS6110-Based Restriction Fragment Length Polymorphism Genotyping of Mycobacterium tuberculosis

  • Robin M. WarrenAffiliated withDST/NRF Centre of Excellence in Biomedical Tuberculosis Research / MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, Stellenbosch University Email author 
  • , Paul D. van HeldenAffiliated withCentre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London, Queen Mary’s School of Medicine and Dentistry
  • , Nicolaas C. Gey van PittiusAffiliated withCentre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London, Queen Mary’s School of Medicine and Dentistry

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Abstract

DNA fingerprinting techniques are based on genome variation and form the basis of molecular epidemiology studies of tuberculosis. A number of markers are in use for the molecular differentiation of Mycobacterium tuberculosis isolates by DNA fingerprinting. One of these markers is the IS6110 insertion element, which may be present in up to 25 copies per M. tuberculosis genome. Variation in both the number and location of the IS6110 elements makes it a very useful marker of strain genotype. IS6110-based DNA fingerprinting is globally considered as the reference genotyping technique for M. tuberculosis isolates. This method is based on visualization of restriction fragment length polymorphisms using a labeled probe derived from IS6110. In this chapter, the method of IS6110 DNA fingerprinting is explained in such a way that it can be easily duplicated by molecular epidemiologists and will give reproducible results.

Keywords

culture DNA extraction ECL labeling IS6110 insertion element RFLP Southern hybridization transposition