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

Publication Type:

Journal Article

Source:

Nucleic Acids Res, Volume 22, Number 20, p.4148-4153 (1994)

DOI Name (links to online publication)

10.1093/nar/22.20.4148

Keywords:

Base Sequence; Cell Line; Chromosomes; Human; Pair 16; Cloning; Molecular; Cosmids; Dna; Exons; Genes; genetics; Globins; Human; Humans; metabolism; methods; Molecular Sequence Data; Nucleic Acid Hybridization; Poly A; Polymerase Chain Reaction; RNA Splicing

Abstract:

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 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

18/01/2013