Advances in Brief Comparative Genomic in Situ Hybridization of Colon Carcinomas with Replication Error '

DNA Preparation and Labeling. Fresh tumor specimens of surgically removed colorectal carcinomas from 18 patients operated at the Department of Surgery,Universityof Regensburg,were snap frozenin liquidnitrogenim mediately after resection and stored at â€”80Â°C until analysis. The mean age of the patients was 65 years (range, 40â€”84years). Whole blood from the same patient was collected as a source of nonneoplastic control DNA. Preparation of genomic DNA was performed by standard methods. Control DNA from leukocytes of the same patient was applied in all experiments. For CGH, tumor and control DNA (1 @g each) were labeled by nick translation in the presence of either digoxigenin-1l-dUTP or biotin-l4-dATP. After determination of DNA concentration, a 10-pi mixture containing equal quantities of 200 ng of each DNA in hybridization solution (50% formamide-300 mM sodium chloride-30 mM sodium citrate-l0% dextrane sulfate) was prepared. In Situ Hybridization. Metaphase chromosomes of normal blood lympho cytes (46,XY) were prepared according to standard protocols of Colcemid arrest,hypotonictreatment,andmethanol:aceticacid(3:1,v/v)fixation.Chro mosome spreads were aged for 70 mm at 95Â°Con a heating block, followed by prehybridization with 10 pi of a mixture of 50% formamide-2X SSC-50 @. tg human Cotl DNA under a 18 X 18-mm coverslip. After incubation at 37Â°C for 2 h, the coverslip was removed, and 10 @tl of hybridizationsolution was applied to the same area and sealed with rubber cement under a coverslip (24 X 24 mm). After hybridization for 3 days at 37Â°C, the preparations were washed 3 times for 5 mm in 0.05X SSC at 42Â°C,and immunodetection was performedas described(10). Microscopic Evaluation. Preparations were embedded in Vectashield anti-bleach medium (Vector Laboratories, Inc., Burlingame, CA) and cx amined in a Zeiss Axioskop fluorescence microscope (Zeiss, Munich, Germany) equipped with a double band pass filter for simultaneous cxci tation of green and red fluorescence. Pictures were recorded on Kodak 400 color slide films and subsequently stored as PCD files on CD-ROM. For evaluation of chromosomal copy number, pictures were evaluated on a PC monitor, and individual chromosomes were identified by diamidino phenylindole banding. Copy number changes of chromosomal regions were apparent by a shift of the chromosomal hybridization image (yellow) toward red (loss) or green (gain). Using this method, the particular chro mosomal regions displaying gains and losses could be unequivocally iden tified. In addition, quantitative analysis of the red:green fluorescence ratio along individual chromosomes was performed using the analySIS (SIS, Muenster, Germany) software package. CGH analysis was performed in dependently by two investigators (J. S. and G. S.) without knowledge of which samples were MIN positive and MIN negative. PCR Analysis. PCR analysis of matched samples of DNA from tumor specimens and blood of the same patient was performed as described previ ously (11). The microsatellite loci were localized on chromosomes Sq (APC), 9 (D9S171),10 (D10S89,Mfd28), lip (P53),and 18q (D18S34,Mfd26). Oligonucleotides used as primers for the PCR were AFM186xc3 for D9S171, APC, Mfd28, P53, and Mfd26; primer sequences have been published previously (11). Abstract