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-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.
Datson
N A
NA
Department of Human Genetics, Leiden University, The Netherlands.
Duyk
G M
GM
Van Ommen
J B
JB
Den Dunnen
J T
JT
D001483:Base Sequence; D002460:Cell Line; D002885:Chromosomes, Human, Pair 16; D003001:Cloning, Molecular / Q000379:methods*; D003360:Cosmids; D005091:Exons; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D014162:Transfection
D011061:Poly A; D005914:Globins