Comparative Genomic Hybridization: DNA Labeling, Hybridization and Detection
Array-CGH involves the comparison of a test to a reference genome using a microarray composed of target sequences with known chromosomal coordinates. The test and reference DNA samples are used as templates to generate two probe DNAs labeled with distinct fluorescent dyes. The two probe DNAs are co-hybridized on a microarray in the presence of Cot-1 DNA to suppress unspecific hybridization of repeat sequences. After slide washes and drying, microarray images are acquired on a laser scanner and fluorescent intensities from every target sequence spot on the array are extracted using dedicated computer programs. Intensity ratios are calculated and normalized to enable data interpretation. Although the protocols explained in this chapter correspond primarily to the use of large-insert clone microarrays in either manual or automated fashion, necessary adaptations for hybridization on microarrays comprising shorter target DNA sequences are also briefly described.
Key wordsProbe labeling random priming hybridization detection comparative genomic hybridization array-CGH
The authors would like to thank Heike Fiegler who developed some of the methods described in this chapter. This work was supported by the Wellcome Trust.
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