Analysis of genomic aberrations using comparative genomic hybridization of metaphase chromosomes

Melanie A. Carless

    Research output: Contribution to journalArticle

    Abstract

    Comparative genomic hybridization (CGH) allows the global screening of copy number aberrations within a sample. Specifically, large (>20 mb) deletions and amplifications are detected, based on utilization of test and reference (karyotypically normal) DNA. These samples are whole-genome amplified by DOPPCR and then differentially labeled with fluorophores via nick translation. Test and reference samples are competitively hybridized to normal metaphase chromosomes. The relative amount of each DNA that binds to a chromosomal locus is indicative of the abundance of that DNA. Thus, if a chromosomal region is amplified, the test DNA will out-compete the reference DNA for binding and fluorescence will indicate amplification. Conversely, if a region is deleted, more reference DNA will bind and fluorescence will indicate a deletion. The following chapter outlines the protocols used for CGH analysis of metaphase chromosomes. These protocols include metaphase chromosome slide preparation, DNA extraction (from blood, cell lines, and microdissected formalin-fixed paraffin-embedded tissue), DOP-PCR, nick translation, in situ hybridization, and fluorescence microscopy and image analysis.

    Original languageEnglish (US)
    Pages (from-to)67-94
    Number of pages28
    JournalMethods in Molecular Biology
    Volume1288
    DOIs
    Publication statusPublished - Jan 1 2015

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    Keywords

    • Comparative genomic hybridization (CGH)
    • Copy number aberrations (CNAs)
    • Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)
    • Fluorescence microscopy
    • Microdissection
    • Nick translation

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

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