Detection of homozygous deletions in tumors by hybridization of representational difference analysis (RDA) products to chromosome-specific YAC clone arrays.
Representational difference analysis (RDA), a subtractive hybridization method that enriches differences between complex genomes, can be used to isolate fragments deleted in tumor genomes. Usually, most of the clones obtained by this approach result from polymorphic fragments. Therefore, identification of homozygously deleted fragments, which can indicate the presence of tumor suppressor loci, is often tedious. To overcome this limitation, we devised a novel strategy in which labeled RDA products are hybridized in toto against membranes spotted with YAC clones covering a region of interest. In such a way, identified YAC clones provide positional information on homozygous deletions and loss of heterozygosity (LOH) regions. We have tested this approach with a tumor known to have a homozygous deletion within a region of LOH on chromosome 13. RDA was performed using representations generated with restriction enzymes Bgl II, Nco I and Xba I, and the difference products of each experiment were separately hybridized to chromosome 13 YAC filters. When collating the map positions of positive YACs from three different RDA experiments a cluster of hits clearly identified the region on chromosome 13 which comprised the homozygous deletion. This shows that our novel approach can be effective.
Zeschnigk
M
M
Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. michael.zeschnigk@uni-essen.de
Horsthemke
B
B
Lohmann
D
D
D001426:Bacterial Proteins; D002872:Chromosome Deletion; D018244:Chromosomes, Artificial, Yeast; D002882:Chromosomes, Human, Pair 13; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm; D015252:Deoxyribonucleases, Type II Site-Specific; D016147:Genes, Tumor Suppressor; D006720:Homozygote; D006801:Humans; D019656:Loss of Heterozygosity; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012175:Retinoblastoma / Q000235:genetics*
D001426:Bacterial Proteins; D004273:DNA, Neoplasm; C052380:endodeoxyribonuclease NcoI; C025580:endodeoxyribonuclease XBAI; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific