COMBO-FISH for focussed fluorescence labelling of gene domains: 3D-analysis of the genome architecture of abl and bcr in human blood cells.
Structural analysis and nanosizing of gene domains requires not only high-resolution microscopy but also improved techniques of fluorescence labelling strongly focussed on the gene domains. To investigate the architecture of abl and bcr in blood cell nuclei forming the Philadelphia chromosome in CML, we applied COMBO-FISH using specifically colocalising combinations of triple strand forming oligonucleotide probes for abl on chromosome 9 and bcr on chromosome 22. Each probe set consisting of 31 homopyrimidine oligonucleotides was computer selected from the human genome database. Measurements by 3D microscopy were compared to results obtained after standard FISH using commercially available abl/bcr BAC probes. The relative radial fluorescence distributions in lymphocyte cell nuclei of healthy donors in comparison to cell nuclei of blood cells of CML patients showed a strong correlation in the location of abl and bcr for both labelling techniques. The absolute distances of the homologous bcr domains and the abl domain-nuclear center-abl domain angles in cell nuclei of CML donors differed significantly from those of healthy donors only when COMBO-FISH was applied. These results indicate that COMBO-FISH may be more sensitive than standard FISH in case of slight modifications in the genome architecture.
Schwarz-Finsterle
Jutta
J
Kirchhoff-Institute of Physics, University of Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany.
Stein
Stefan
S
Grossmann
Constance
C
Schmitt
Eberhard
E
Schneider
Helmut
H
Trakhtenbrot
Luba
L
Rechavi
Gideon
G
Amariglio
Ninette
N
Cremer
Christoph
C
Hausmann
Michael
M
D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000097:blood*; D016313:Genes, abl; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010677:Philadelphia Chromosome; D051562:Proto-Oncogene Proteins c-bcr / Q000097:blood / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D014178:Translocation, Genetic
D016044:Fusion Proteins, bcr-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr