Isolation of transcribed sequences from human 17q by magnetic bead capture
- Univ. of Michigan, Ann Arbor (United States); and others
The identification and characterization of transcribed sequences from large genomic regions is a crucial aim of current genome research. A number of novel techniques have been introduced that target the difficult problem of identifying low abundance coding sequences in large cloned DNA segments. A particularly promising approach is magnetic bead capture of cDNA fragments. As part of our study to identify genes from the long arm of human chromosome 17 and homologous regions of mouse chromosome 11, we have applied this technology to the region surrounding the EPB3 locus at 17q21.3. Cosmid clones, derived from screening the Los Alamos chromosome 17 gridded cosmid library with inter-Alu PCR products from two overlapping EPB3{sup +} YACs, were used as hybridization reagents together with a control genomic clone containing the PGK-1 locus. Linkered cosmid clones were PCR-amplified and hybridized with amplified cDNA fragments from human fetal brain and hippocampus libraries (Stratagene). Following magnetic bead capture, specifically bound cDNAs were eluted, amplified and cloned into pAMP-1 (Stratagene) or M13 vectors. cDNAs originating from the genomic clones of origin were identified by hybridization with cosmid insert DNA. Following two rounds of selection, approximately 30% of captured cDNA clones hybridized with the starting cosmids and all of these clones mapped back to the target region. Beginning with five cosmid clones, 10 different cDNAs clones, ranging in size from 200 to 600 bp, were identified. Sequence analysis of the cDNAs and database searches have shown the identification of novel cDNA species originating from the region. Our eventual goals are to contribute to the identification of the estimated 2000 genes in a 60 Mb region of human chromosome 17q and homologous regions of mouse chromosome 11 and provide a direct species comparison between the human and mouse genomes over large conserved regions.
- OSTI ID:
- 134794
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1533
- 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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Related Subjects
BASIC STUDIES
44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
GENES
GENETIC MAPPING
HUMAN CHROMOSOME 17
GENETICS
COMPARATIVE EVALUATIONS
SCREENING
DNA
DNA HYBRIDIZATION
DNA-CLONING
DNA SEQUENCING
FRAGMENTATION
MICE
CHROMOSOMES
TRANSCRIPTION
YEASTS
POLYMERASE CHAIN REACTION