Abstract
Typical CRISPR (clustered, regularly interspaced, short palindromic repeat) regions are constituted by short direct repeats (DRs), interspersed with similarly sized non-repetitive spacers, derived from transmissible genetic elements, acquired when the cell is challenged with foreign DNA. The analysis of the structure, in number and nature, of CRISPR spacers is a valuable tool for molecular typing since these loci are polymorphic among strains, originating characteristic signatures. The existence of CRISPR structures in the genome of the members of Mycobacterium tuberculosis complex (MTBC) enabled the development of a genotyping method, based on the analysis of the presence or absence of 43 oligonucleotide spacers separated by conserved DRs. This method, called spoligotyping, consists on PCR amplification of the DR chromosomal region and recognition after hybridization of the spacers that are present. The workflow beneath this methodology implies that the PCR products are brought onto a membrane containing synthetic oligonucleotides that have complementary sequences to the spacer sequences. Lack of hybridization of the PCR products to a specific oligonucleotide sequence indicates absence of the correspondent spacer sequence in the examined strain. Spoligotyping gained great notoriety as a robust identification and typing tool for members of MTBC, enabling multiple epidemiological studies on human and animal tuberculosis.
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Acknowledgments
This work was supported by Fundacão para a Ciência e Tecnologia (FCT) through project PTDC/CVT/117794/2010 and in the framework of Projecto 3599—Promover a Produção Científica e Desenvolvimento Tecnológico e a Constituição de Redes Temáticas.
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Botelho, A., Canto, A., Leão, C., Cunha, M.V. (2015). Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) Analysis of Members of the Mycobacterium tuberculosis Complex. In: Cunha, M., Inácio, J. (eds) Veterinary Infection Biology: Molecular Diagnostics and High-Throughput Strategies. Methods in Molecular Biology, vol 1247. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2004-4_27
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DOI: https://doi.org/10.1007/978-1-4939-2004-4_27
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