The long term goal of this project is to develop a method to identify rapidly individual transcription units in the human genome using a 3'- terminal exon trapping strategy developed during the first period of this award. This strategy utilizes a mammalian expression vector containing an incomplete transcription unit lacking a 3'-terminal exon. Only in the presence of inserted genomic DNA does the vector produce stable poly(A)+RNA upon transfection of cultured cells. RNA, and thereby the gene from which it originated, is detected and obtained by RT/PCR amplification. In the next five years we plan to: 1) Refine the technique. We will explore technique modifications designed to minimize false positives an maximize sensitivity. We will also develop an in vitro trapping technique. 2) Extend trapping to more complex sources of DNA. We have just trapped our first exons rom cosmids. We wish to extend this analysis, with an emphasis on determining technique efficiency when applied to cosmid and YAC DNA. 3) Identify potential trnscription units within a specified chromosomal region. To test the efficiency and sensitivity of our exon trapping protocol we plan to identify transcriptional units within the p22 and p23 regions of human chromosome 6. 4) Creation of X chromosome specific libraries. Using an arrayed library of X-specific cosmids and YACs presently available in the Baylor Genome Center as sources of genomic DNA, we plan to isolate X chromosome 3'- terminal exons. Isolated exons will be sequenced, catalogued, and arrayed for use by other investigators.
| Krizman, D B; Berget, S M (1993) Efficient selection of 3'-terminal exons from vertebrate DNA. Nucleic Acids Res 21:5198-202 |
