Isolation of a cosmid sublibrary for a region of chromosome 12 frequently amplified in human cancers using a complex chromosome microdissection probe.
Chromosome-specific cosmid libraries are an extremely useful resource for positional cloning projects. Once a particular region of interest has been identified, it would be of value to have an approach for isolating chromosome band-specific cosmids that could be assembled into a sublibrary for rapid screening. We constructed a region-specific sublibrary of 700 cosmids by screening a chromosome 12-specific cosmid library with a complex probe generated by degenerate oligonucleotide-primed PCR of a microdissected homogeneously staining region containing sequences amplified from chromosome 12q13-q15. Based on fluorescence in situ hybridization, approximately 60% of the cosmids in the sublibrary were derived from the microdissected region. To demonstrate further the utility of this sublibrary, a 150-kb contig containing the SAS and CDK4 genes was constructed, as well as several additional contigs between CDK4 and MDM2. This study demonstrates the possibility of utilizing probes generated by microdissection for assembling band-specific sublibraries that are amenable to rapid screening with multiple markers.
Elkahloun
A G
AG
Laboratory of Cancer Genetics, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Meltzer
P S
PS
Guan
X Y
XY
McNinch
J S
JS
Trent
J M
JM
de Jong
P J
PJ
D001483:Base Sequence; D002874:Chromosome Mapping; D002881:Chromosomes, Human, Pair 12; D003360:Cosmids; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D017931:DNA Primers; D005784:Gene Amplification; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D011518:Proto-Oncogene Proteins
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases