E. Gracia et al., ISOLATION OF GENES AMPLIFIED IN HUMAN CANCERS BY MICRODISSECTION MEDIATED CDNA CAPTURE, Human molecular genetics, 5(5), 1996, pp. 595-600
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.