An integrated approach for identifying and mapping human genes
- Albert Einstein College of Medicine, Bronx, NY (United States)
- Yale Univ. School of Medicine, New Haven, CT (United States)
The authors have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. The authors describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and they present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, they obtained the sequence, including an intron-exon junction, flanking a particular truncation event. 24 refs., 5 figs., 1 tab.
- OSTI ID:
- 6247377
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 90:10; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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