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

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 a-globin gene cluster on chromosome 16. The 3'-terminal exons of the human Ca1-, ¢2's and 0-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the a1-globin gene. This exon appears to belong to a previously unidentified gene within the a-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.

RNA Aptamers Targeting the Carboxyl Terminus of KRAS Oncoprotein Generated by an Improved SELEX with Isothermal RNA Amplification

“…The RNA amplification protocol was basically according to the NASBA method (Datson et al, 1994;Malek et al, 1994;Oehlenschlager et al, 1996) with the following modifications. In the NASBA method, complementary RNAs are amplified from a 3Ј primer that is attached to the T7 promoter sequence.…”