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Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes

Nature Protocols volume 2, pages 577–587 (2007)Cite this article

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Abstract

Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40–200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.

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Figure 1: Principle of array-CGH.
Figure 2: Schematic overview of target DNA preparation for array production (a) DOP-PCR: The variation of six consecutive bases within the primer (N) and the low annealing temperature during the first amplification cycles allow the primers to bind along the template DNA.
Figure 3: Distinct banding patterns of DOP-PCR and amino-linking PCR products using long-insert genomic clones as templates.
Figure 4: Chromosome 10 array-CGH profile of a colon cancer cell line on a whole-genome tiling path microarray.

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Change history

  • 28 June 2007

    In the version of this article originally published online, the author omitted to acknowledge the contributions of the following parties to this article: The protocols described in Steps 1-9 were originally developed by Sean Humphray and members of the Wellcome Trust Sanger Institute Core Mapping Group; Steps 15–27 were originally developed or are modified from protocols developed by David Vetrie, Cordelia Langford and other members of the Wellcome Trust Sanger Institute Microarray Facility. The original protocols are available at http://www.sanger.ac.uk/Projects/Microarrays/arraylab/protocol3b.pdf and http://www.sanger.ac.uk/Projects/Microarrays/arraylab/protocol4.pdf. The authors are supported by the Wellcome Trust. This error has been corrected in the PDF version of the article.

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Authors and Affiliations

  1. The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK

    Heike Fiegler, Richard Redon & Nigel P Carter

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  1. Heike Fiegler
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  2. Richard Redon
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  3. Nigel P Carter
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Correspondence to Heike Fiegler.

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Fiegler, H., Redon, R. & Carter, N. Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes. Nat Protoc 2, 577–587 (2007). https://doi.org/10.1038/nprot.2007.53

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