CRISPR pp 111-131 | Cite as

Spacer-Based Macroarrays for CRISPR Genotyping

  • Igor MokrousovEmail author
  • Nalin Rastogi
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)


Macroarray-based analysis is a powerful and economic format to study variations in “clustered regularly interspaced short palindromic repeat (CRISPR)” loci in bacteria. To date, it was used almost exclusively for Mycobacterium tuberculosis and was named spoligotyping (spacer oligonucleotides typing). Here, we describe the pipeline of this approach that includes search of loci and selection of spacers, preparation of the membrane with immobilized probes and spoligotyping itself (PCR and reverse hybridization).

Key words

Spoligotyping Reverse hybridization Macroarrays 



We are grateful to Thierry Zozio, Elisabeth Streit and Julie Millet (Institut Pasteur de la Guadeloupe), Anna Vyazovaya and Olga Narvskaya (St. Petersburg Pasteur Institute) for helpful discussions. Igor Mokrousov gratefully acknowledges support from Russian Science Foundation (project 14-14-00292).


  1. 1.
    Hermans PWM, van Soolingen D, Bik EM, de Haas PEW, Dale JW, van Embden JDA (1991) The insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun 59:2695–2705PubMedCentralPubMedGoogle Scholar
  2. 2.
    Groenen PM, Bunschoten AE, van Soolingen D, van Embden JD (1995) Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol Microbiol 10:1057–1065CrossRefGoogle Scholar
  3. 3.
    Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S et al (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35:907–914PubMedCentralPubMedGoogle Scholar
  4. 4.
    Kremer K, Bunschoten A, Schouls L, van Soolingen D, and van Embden J (2002) Spoligotyping—a PCR-based method to simultaneously detect and type Mycobacterium tuberculosis complex bacteria, version 4, 11/8/02.
  5. 5.
    Demay C, Liens B, Burguière T, Hill V, Couvin D, Millet J et al (2012) SITVITWEB—a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology. Infect Genet Evol 12:755–766CrossRefPubMedGoogle Scholar
  6. 6.
    van Embden JDA, van Gorkom T, Kremer K, Jansen T, van der Zeijst BAM, Schouls LM (2000) Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria. J Bacteriol 182:2393–2401CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    van der Zanden AG, Kremer K, Schouls LM (2002) Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides. J Clin Microbiol 40:4628–4639CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Brudey K, Gutierrez MC, Vincent V, Parsons LM, Salfinger M, Rastogi N et al (2004) Mycobacterium africanum genotyping using novel spacer oligonucleotides in the direct repeat locus. J Clin Microbiol 42:5053–5057CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Javed MT, Aranaz A, de Juan L, Bezos J, Romero B, Alvarez J et al (2007) Improvement of spoligotyping with additional spacer sequences for characterization of Mycobacterium bovis and M. caprae isolates from Spain. Tuberculosis 87:437–445CrossRefPubMedGoogle Scholar
  10. 10.
    Zhang J, Abadia E, Refregier G, Tafaj S, Boschiroli ML, Guillard B, Andremont A et al (2010) Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of “spoligotyping” with new spacers and a microbead-based hybridization assay. J Med Microbiol 59:285–294CrossRefPubMedGoogle Scholar
  11. 11.
    Sola C, Filliol I, Legrand E, Lesjean S, Locht C, Supply P et al (2003) Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics. Infect Genet Evol 3:125–133CrossRefPubMedGoogle Scholar
  12. 12.
    Jansen R, Embden JD, Gaastra W, Schouls LM (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43:1565–1575CrossRefPubMedGoogle Scholar
  13. 13.
    Mokrousov I, Narvskaya O, Limeschenko E, Vyazovaya A (2005) Efficient discrimination within a Corynebacterium diphtheriae epidemic clonal group by a novel macroarray-based method. J Clin Microbiol 43:1662–1668CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Mokrousov I, Limeschenko E, Vyazovaya A, Narvskaya O (2007) Corynebacterium diphtheriae spoligotyping based on combined use of two CRISPR loci. Biotechnol J 2:901–906CrossRefPubMedGoogle Scholar
  15. 15.
    Shariat N, Dudley EG (2014) CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 80(2):430–439. doi: 10.1128/AEM. 02790-13 CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Grissa I, Vergnaud G, Pourcel C (2007) CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 35:W52–57CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27:573–580CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Dale JW, Brittain D, Cataldi AA, Cousins D, Crawford JT, Driscoll J et al (2001) Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardised nomenclature. Int J Tuberc Lung Dis 5:216–219PubMedGoogle Scholar
  19. 19.
    Tang C, Reyes JF, Luciani F, Francis AR, Tanaka MM (2008) spolTools: online utilities for analyzing spoligotypes of the Mycobacterium tuberculosis complex. Bioinformatics 24:2414–2415CrossRefPubMedGoogle Scholar
  20. 20.
    Reyes JF, Francis AR, Tanaka MM (2008) Models of deletion for visualizing bacterial variation: an application to tuberculosis spoligotypes. BMC Bioinformatics 9:496CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Ellson J, Gansner E, Koutsofios L, North SC, Woodhull G (2002) Graphviz—open source graph drawing tools (Mutzel P, Jünger M, Leipert S, eds.). Springer-Verlag Berlin, Heidelberg, pp 483–484Google Scholar
  22. 22.
    Groenheit R, Ghebremichael S, Pennhag A, Jonsson J, Hoffner S, Couvin D et al (2012) Mycobacterium tuberculosis strains potentially involved in the TB epidemic in Sweden a century ago. PLoS One 7:e46848CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Grimont PAD (2000) Taxotron package. Taxolab, Institut Pasteur, ParisGoogle Scholar
  24. 24.
    Coll F, Mallard K, Preston MD, Bentley S, Parkhill J, McNerney R et al (2012) SpolPred: rapid and accurate prediction of Mycobacterium tuberculosis spoligotypes from short genomic sequences. Bioinformatics 28:2991–2993CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chencik A (1994) TaqStart antibody: “hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16:1134–1137PubMedGoogle Scholar
  26. 26.
    Molhuizen HO, Bunschoten AE, Schouls LM, van Embden JD (1998) Rapid detection and simultaneous strain differentiation of Mycobacterium tuberculosis complex bacteria by spoligotyping. Methods Mol Biol 101:381–394PubMedGoogle Scholar
  27. 27.
    Van Der Zanden AG, Te Koppele-Vije EM, Vijaya Bhanu N, Van Soolingen D, Schouls LM (2003) Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis. J Clin Microbiol 41:1101–1108CrossRefGoogle Scholar
  28. 28.
    Lazzarini LC, Rosenfeld J, Huard RC, Hill V, Lapa e Silva JR, DeSalle R et al (2012) Mycobacterium tuberculosis spoligotypes that may derive from mixed strain infections are revealed by a novel computational approach. Infect Genet Evol 12:798–806CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratory of Molecular MicrobiologySt. Petersburg Pasteur InstituteSt. PetersburgRussia
  2. 2.WHO Supranational TB Reference Laboratory, Tuberculosis & Mycobacteria UnitInstitut Pasteur de la GuadeloupeAbymesFrance

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