Isolation of Coding Sequences from Yeast Artificial Chromosome (Yac): Clones by Exon Amplification
互联网
514
Exon amplification is a technique designed to address a central problem in mammalian molecular genetics—how to extract coding sequences from large tracts of genomic DNA. As shown in Fig. 1 , the technique (also known as exon trapping) exploits the ability of the eukaryotic splicing machinery to detect splice sites flanking exon sequences in pre-mRNA molecules. The original exon trapping vector pSPL1 developed by Buckler et al. (1 ) and its subsequently improved derivative pSPL3 (2 ) allow segments of genomic DNA to be cloned into an HIV-tat intron that is flanked by the 5′ and 3′ splice sites and exons of the viral gene. Recombinant clones are transfected into COS-7 cells, which support high levels of transcription driven by the SV40 early promoter of the vector. During in vivo splicing, the 5′ and 3′ splice sites flanking an exon contained within the genomic insert are paired with the HIV-tat splice sites, with the result that the genomic exon is retained in the mature cytoplasmic poly A+ RNA. Reverse transcription of the cytoplasmic RNA is followed by PCR using primers specific for the HIV-tat exons to amplify the “trapped exon.“ After a secondary (nested) PCR amplification, the PCR products are cloned into a suitable plasmid vector.
Fig. 1. An overview of exon amplification. ss, splice site. SD6 and SD2, primary PCR primers. dUSD2 and dUSA4, secondary PCR primers. SD6 and dUSD2 are the sense primers. Numbers below boxes indicate the number of bases from the first nucleotide of the primer to the 3′ terminus of the vector exon. Note that the secondary PCR primers are nested (internal to the primary PCR primers). See text for details.
