An integrated approach for identifying and mapping human genes

R. Das Gupta, B. Morrow, I. Marondel, S. Parimoo, V. L. Goei, J. Gruen, S. Weissman, A. Skoultchi, R. Kucherlapati

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

We 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. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.

Original languageEnglish (US)
Pages (from-to)4364-4368
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number10
StatePublished - Jan 1 1993
Externally publishedYes

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Keywords

  • cDNA libraries
  • expressed sequence tags
  • homologous recombination
  • human gene mapping
  • yeast artificial chromosomes

ASJC Scopus subject areas

  • General

Cite this

Das Gupta, R., Morrow, B., Marondel, I., Parimoo, S., Goei, V. L., Gruen, J., Weissman, S., Skoultchi, A., & Kucherlapati, R. (1993). An integrated approach for identifying and mapping human genes. Proceedings of the National Academy of Sciences of the United States of America, 90(10), 4364-4368.