Molecular cytogenetics: unraveling of the genetic composition of individual cells by fluorescence in situ hybridization and digital imaging microscopy.
Molecular biology techniques allow the unraveling of the genetic alterations that cause or accompany malignant disease. Since tumors are often heterogeneous, biochemical analysis of tissue homogenates is of limited diagnostic value. This paper gives examples of methods that are presently operational to analyze the genetic composition of individual cells. They are based on fluorescence in situ hybridization (FISH) and digital imaging microscopy. First, the current status of indirect and direct FISH staining methods with respect to probe labeling, detection sensitivity, multiplicity, and DNA resolution is summarized. Microscope hardware as well as charge-coupled device (CCD) cameras required for FISH analysis are then described. Applications potentially important for the analysis of urological malignancies, such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells) and high-resolution DNA mapping on highly extended chromatin, are described in detail. Finally, the limitations of the present methodology and its future prospects are discussed.
Tanke
H J
HJ
Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands.
Florijn
R J
RJ
Vrolijk
J
J
Raap
A K
AK
D002874:Chromosome Mapping; D003582:Cytogenetics / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000235:genetics*; D012815:Signal Processing, Computer-Assisted