ISOLATION OF GENES AMPLIFIED IN HUMAN CANCERS BY MICRODISSECTION MEDIATED CDNA CAPTURE

It has been increasingly recognized that homogeneously staining region s (hsr) in human cancers may be complex structures composed of large a mplified DNA domains containing multiple genes, It is therefore import ant to devise strategies for the rapid isolation of cDNAs expressed fr om these structures, Using a procedure we term microdissection mediate d cDNA capture, we recovered hsr specific cDNAs from two different hum an tumors. The glioblastoma cell line TX3868 and the human sarcoma cel l line OsA-CL carry hsrs containing amplified sequences from chromosom e 12q13-15. We recovered 17 hsr specific cDNAs following microdissecti on of these hsrs which had been previously hybridized in situ with lin kered cDNA. Northern blot analysis with these cDNAs revealed hybridiza tion to distinct transcripts in OsA-CL RNA and TX3868 RNA, None of the OsA-CL cDNA clones showed cross hybridization with the TX3868 cDNAs s uggesting 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 der ived from this region and the utility of microdissection mediated cDNA capture to gain rapid access to cDNAs transcribed from amplified gene s.