Identification of 3'-terminal exons from yeast artificial chromosomes.

  1. D B Krizman,
  2. T A Hofmann,
  3. U DeSilva,
  4. E D Green,
  5. P S Meltzer, and
  6. J M Trent
  1. Laboratory of Cancer Genetics, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.

Abstract

We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.

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