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Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes

Nature Protocols volume 3, pages 849–865 (2008)Cite this article

Abstract

High-throughput genome-wide screening methods to detect subtle genomic imbalances are extremely important for diagnostic genetics and genomics. Here, we provide a detailed protocol for a microarray-based technique, applying the principle of multiplex amplifiable probe hybridization (MAPH). Methodology and software have been developed for designing unique PCR-amplifiable sequences (400–600 bp) covering any genomic region of interest. These sequences are amplified, cloned and spotted onto arrays (targets). A mixture of the same sequences (probes) is hybridized to genomic DNA immobilized on a membrane. Bound probes are recovered and quantitatively amplified by PCR, labeled and hybridized to the array. The procedure can be completed in 4–5 working days, excluding microarray preparation. Unlike array-comparative genomic hybridization (array-CGH), test DNA of specifically reduced complexity is hybridized to an array of identical small amplifiable target sequences, resulting in increased hybridization specificity and higher potential for increasing resolution. Array-MAPH can be used for detection of small-scale copy-number changes in complex genomes, leading to genotype–phenotype correlations and the discovery of new genes.

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Figure 1: A flow diagram outlining the steps involved in array-multiplex amplifiable probe hybridization (MAPH) methodology.
Figure 2: Agarose gel electrophoresis for quality control of probe amplification with specific primers.
Figure 3: Agarose gel electrophoresis for cloning efficiency control, performed by insert amplification with PZA and PZB primers from bacterial colonies.
Figure 4: Probe amplification with PZA and PZB primers from hybridized filters.
Figure 5: Scanned images of successful and unsuccessful array-multiplex amplifiable probe hybridization (MAPH) reactions.
Figure 6: Examples of array-multiplex amplifiable probe hybridization (MAPH) profiles for normal DNA samples.
Figure 7: Array-multiplex amplifiable probe hybridization (MAPH) profile of patient A-2879 carrying a known duplication of Xp22.32-p22.31.
Figure 8: Array-multiplex amplifiable probe hybridization (MAPH) profile of patient 22467 carrying a deletion of 1.5 mb on chromosome X.
Figure 9: Array-multiplex amplifiable probe hybridization (MAPH) profile of patient A045 resulted in the detection of a 500-kb deletion on Xq25, based on a single-probe deviation.

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Acknowledgements

This work was funded by the grants 30/2001 from the Cyprus RPF, QLRT-2001-01810 from the EURO-MRX EU, 5467 from the Estonian Science Foundation, 0182649s04 and PBGMR06907 from the Estonian Ministry of Education and Research and 070191/Z/03/Z from the Wellcome Trust International Senior Research Grant. We also thank D. Andreou, C. Tryfonos, E. Hadjiyanni, C. Pitta, C. Antoniades, S. Bashiardes and G. Slavin for their contribution. Many thanks to Prof. J. Vermeesch, Dr. K. Õunap and Dr. R. Ordania for the provision of DNA samples and the Wellcome Trust Sanger Institute for the provision of BAC/PAC clones.

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Authors and Affiliations

  1. Department of Cytogenetics, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus

    Ludmila Kousoulidou, Carolina Sismani & Philippos C Patsalis

  2. Biotechnology Department, Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia

    Katrin Männik, Olga Žilina, Sven Parkel, Helen Puusepp, Neeme Tõnisson, Priit Palta, Maido Remm & Ants Kurg

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Contributions

L.K. and K.M. contributed equally to this work. A.K. and P.C.P. are the principal investigators of this study.

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Correspondence to Philippos C Patsalis.

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Kousoulidou, L., Männik, K., Sismani, C. et al. Array-MAPH: a methodology for the detection of locus copy-number changes in complex genomes. Nat Protoc 3, 849–865 (2008). https://doi.org/10.1038/nprot.2008.49

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