A rapid method for isolation chromosome band specific cDNAs and genomic cDNAs with microdissection/microcloning libraries
- Northwestern Univ. Medical School, Chicago, IL (United States)
Positional cloning provides a powerful tool for isolation of the gene(s) whose location has been identified while whose biochemical product and function are unknown. Previous techniques for positional cloning require construction of the DNA contig either with YAC, cosmid or bacteriophage clones, which is time consuming. To facilitate this process, we developed a rapid method in which microdissection/microcloning DNA libraries are used as probe pools to directly screen genomic DNA and cDNA libraries. In our initial experiment, we used chromosome band-specific libraries from 5q13 and 2q33 constructed with our microdissection techniques as probe pools to direct screen human genomic library and human brainstem or frontal cortex cDNA libraries using sonicated total human genomic DNA as the competitor. Five genomic DNA clones and 2 cDNA clones have been isolated with the 5q13 library and 11 cDNA clones have been isolated with the 2q33 library. Characterization of 3 genomic and 4 cDNA clones with fluorescence in situ hybridization indicated that these clones are derived from 5q13 and 2q33, respectively. One of our genomic clones had hybridization signals not only at 5q13 but also at 5p13, 5q21 and 5q31, indicating that existence of chromosome-specific repetitive sequences in human chromosome 5. One cDNA clone isolated with 2q33 library contains a polymorphic dinucleotide repeat. Linkage analysis showed that this clone has close linkage with the ALS2 locus previously mapped close to D2S72/D2S116.
- OSTI ID:
- 134416
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1150
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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