A direct method for the positional cloning of cDNAs by in situ hybridization and microdissection of metaphase chromosomes
- Harvard Medical School, Boston, MA (United States)
We have developed a novel strategy for the expedited cloning of cDNAs for genes contained within defined cytogenetic regions of the genome. A cDNA library is prepared by RT-PCR of RNA using primers with 3{prime} to 5{prime} sequences consisting of 4 specific nucleotides, followed by 5 random nucleotides and then 20 specific nucleotides. This library is hybridized to acid-fixed metaphase chromosomes immobilized on glass over slips. The chromosomal region of interest is microdissected, and the cDNAs hybridized to the dissected chromosomal fragments are amplified by PCR using primers corresponding to the specific 20-nucleotide sequence present at the 5{prime} end of the primers utilized to generate the original cDNA library. The PCR products obtained can then be screened for genetic alterations, such as point mutations, rearrangements, deletions, or amplification. This strategy has been tested by cloning cDNAs from double minute chromosomes of the colonic carcinoma cell line Colo320, which is known to possess amplified copies of the MYC and PVT genes. Microdissection and PCR of small numbers of double minute chromosomes (5-15) has yielded cDNAs containing sequences of both of these genes as well as several other previously unknown but presumably linked genes amplified in the Colo320 line. This strategy appears to offer a rapid and general method for the isolation of DNA for candidate genes which lie at the positions of various cytogenetic abnormalities or which map to particular regions within the genome.
- OSTI ID:
- 134478
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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