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Nucleic Acids Research, 1994, Vol. 22, No. 20 4148-4153
© 1994

MOLECULAR BIOLOGY

Specific isolation of 3'-terminal exons of human genes by exon trapping

Nicole A. Datson, Geoffrey M. Duyk1, Gert-Jan B. van Ommen and Johan T. Den Dunnen*

Department of Human Genetics, Medical Genetics Centre-South West Netherlands, Leiden University Wassenaarseweg 72, 2333 Al Leiden, The Netherlands 1Department of Genetics, Harvard Medical School 200 Longwood Avenue, Boston, MA 02115, USA

*To whom correspondence should be addressed

Received July 1, 1994. Revised September 25, 1994. Accepted September 25, 1994.

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3'-terminal exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' exons in addition to a great reduction of non-specific background. As a test for the system, 3' exon trapping was performed using a cosmid containing the A-globin gene cluster on chromosome 16. The 3'-terminal exons of the human A1, Z2. and {theta}x-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the a-globin gene. This exon appears to belong to a previously unidentified gene within the {alpha}-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.


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