SPECIFIC ISOLATION OF 3'-TERMINAL EXONS OF HUMAN GENES BY EXON TRAPPING

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-SDP 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 alpha-globin gene cluster on chromosome 16. The 3'-terminal exons of the human alpha(1)-, zeta(2)-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha(1)-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.