TY - JOUR
T1 - Analysis of genomic aberrations using comparative genomic hybridization of metaphase chromosomes
AU - Carless, Melanie A.
N1 - Publisher Copyright:
© Springer Science+Business Media New York 2015.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - Comparative genomic hybridization (CGH)
KW - Copy number aberrations (CNAs)
KW - Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)
KW - Fluorescence microscopy
KW - Microdissection
KW - Nick translation
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U2 - 10.1007/978-1-4939-2474-5_6
DO - 10.1007/978-1-4939-2474-5_6
M3 - Article
C2 - 25827876
AN - SCOPUS:84926666101
VL - 1288
SP - 67
EP - 94
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
SN - 1064-3745
ER -