Isolation of genes amplified in human cancers by microdissection mediated cDNA capture.
It has been increasingly recognized that homogeneously staining regions (hsr) in human cancers may be complex structures composed of large amplified DNA domains containing multiple genes. It is therefore important to devise strategies for the rapid isolation of cDNAs expressed from these structures. Using a procedure we term microdissection mediated cDNA capture, we recovered hsr specific cDNAs from two different human tumors. The glioblastoma cell line TX3868 and the human sarcoma cell line OsA-CL carry hsrs containing amplified sequences from chromosome 12q13-15. We recovered 17 hsr specific cDNAs following microdissection of these hsrs which had been previously hybridized in situ with linkered cDNA. Northern blot analysis with these cDNAs revealed hybridization to distinct transcripts in OsA-CL RNA and TX3868 RNA. None of the OsA-CL cDNA clones showed cross hybridization with the TX3868 cDNAs suggesting that despite their coincident band localization on 12q, the OsA-CL and TX3868 amplification units do not completely overlap. These results significantly increase the number of amplified genes assigned to the 12q13-15 amplicon illustrating both the complexity of hsrs derived from this region and the utility of microdissection mediated cDNA capture to gain rapid access to cDNAs transcribed from amplified genes.
Gracia
E
E
Laboratory of Cancer Genetics, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Fischer
U
U
elKahloun
A
A
Trent
J M
JM
Meese
E
E
Meltzer
P S
PS
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D018076:DNA, Complementary / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D005909:Glioblastoma / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009369:Neoplasms / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D012516:Osteosarcoma / Q000235:genetics; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004273:DNA, Neoplasm