Increased exon-trapping efficiency through modifications to the pSPL3 splicing vector.
Exon trapping allows for the rapid identification and cloning of coding regions from cloned eukaryotic DNA. In preliminary experiments, we observed two phenomena which limited the exon-trapping efficiency of pSPL3-based systems. The first factor that affected performance was revealed when we found that up to 50% of the putative trapped exons contained sequences derived from the intron of the pSPL3 trapping vector. Removal of the DNA sequences responsible for the cryptic splice event from the original splicing vector resulted in a new vector, pSPL3B. We demonstrate that pSPL3B virtually eliminates pSPL3-only spliced products while maximizing the proportion of exon traps containing genomic DNA (> 98%). The other step which impacted performance was our observation that a majority of the ampicillin-resistant (APR) clones produced after shotgun subcloning from ApR cosmids into pSPL3 were untrappable, pSPL3-deficient, recircularized cosmid vector fragments. Replacement of the pSPL3 ApR gene with the CmR cassette encoding chloramphenicol (Cm) acetyltransferase enabled selection for only pSPL3-containing CmR clones. We show a 30-40-fold increase in the initial subcloning efficiency of cosmid-derived fragments with pSPL3-CAM, when compared to pSPL3. The collective vector alterations described improve the overall exon-trapping efficiency of the pSPL3-based trapping system.
Burn
T C
TC
Department of Human Genetics, Integrated Genetics Inc., Framingham, MA 01701-9332, USA.
Connors
T D
TD
Klinger
K W
KW
Landes
G M
GM
D001483:Base Sequence; D002885:Chromosomes, Human, Pair 16; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant; D005091:Exons; D015756:Genes, tat; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data
D017931:DNA Primers; D004274:DNA, Recombinant; D004247:DNA