Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors.
Comparative genomic hybridization (CGH) is a powerful new method for molecular cytogenetic analysis of cancer. In a single hybridization, CGH provides an overview of DNA sequence copy number changes (losses, deletions, gains, amplifications) in a tumor specimen and maps these changes on normal chromosomes. CGH is based on the in situ hybridization of differentially labeled total genomic tumor DNA and normal reference DNA to normal human metaphase chromosomes. After hybridization and fluorescent staining of the bound DNAs, copy number variations among the different sequences in the tumor DNA are detected by measuring the tumor/normal fluorescence intensity ratio for each locus in the target metaphase chromosomes. CGH is in particular useful for analysis of DNA sequence copy number changes in common solid tumors where high-quality metaphase preparations are often difficult to make, and where complex karyotypes with numerous markers, double minutes, and homogeneously stained chromosomal regions are common. CGH only detects changes that are present in a substantial proportion of tumor cells (i.e., clonal aberrations). It does not reveal translocations, inversions, and other aberrations that do not change copy number. At present, CGH is a research tool that complements previous methods for genetic analysis. CGH will advance our understanding of the genetic progression of cancer and highlight important genomic regions for further study. Direct clinical applications of CGH are possible, but will require further development and validation of the technique. We describe here our recent optimized procedures for CGH, including DNA labeling, hybridization, fluorescence microscopy, digital image analysis, data interpretation, and quality control, emphasizing those steps that are most critical. We will also assess sensitivity and resolution limits of CGH as well as discuss possible future technical improvements.
Kallioniemi
O P
OP
Department of Laboratory Medicine, Tampere University Hospital.
Kallioniemi
A
A
Piper
J
J
Isola
J
J
Waldman
F M
FM
Gray
J W
JW
Pinkel
D
D
D002869:Chromosome Aberrations; D002874:Chromosome Mapping; D004273:DNA, Neoplasm / Q000032:analysis*; D016678:Genome; D006801:Humans; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D012680:Sensitivity and Specificity
D004273:DNA, Neoplasm