Inverse-PCR Screen (SLIP)
Directed Approach for Isolating cDNAs
Mapping of ESTs to gene annotations shows that there are 3,125 annotated protein-coding genes not yet represented in the DGC. In addition, the genome sequence annotation predicts that approximately 20% of Drosophila genes produce two or more alternatively spliced transcripts. Thus, at least 2,500 annotated alternatively spliced protein-coding transcripts are also not yet represented in the DGC. Sequencing of the most recent 10,000 5' ESTs identified cDNAs for only 96 additional genes (1% yield). Although EST sequencing of new libraries from different tissues and developmental stages might marginally increase the rate at which additional genes are sampled by EST sequencing, an efficient method for directed screening of cDNA libraries to recover clones for specific transcripts would be very useful.
We have devised a screening method called Self-Ligation of Inverse PCR Products or SLIP. SLIP is a rapid and efficient method for plasmid library screening that can recover full-length cDNAs representing relatively rare and alternatively spliced transcripts of interest. Results from an initial pilot experiment performed on 153 transcription factor target genes identified 82 full-length cDNA clones. These results justified a larger scale screen which has been completed for the missing genes in our collection. Approximately 4,397 clones representing 1,768 genes have been chosen for further sequencing which are now in our full-insert sequencing pipeline.
To date, we have submitted data from this screen to GenBank for 1490 clones representing 981 genes. All clones from this screen are available at the DGRC.
Please NOTE: Clones isolated from this screening process have been PCR amplified using a mixture of 4 different plasmid cDNA libraries as template: GH, LD, LP, and SD cDNA libraries(see our FAQ for more info about these libraries). Clones from this screen cannot be used to measure tissue specific gene expression levels since both the tissue/stage information is irrelevant due to the combination of PCR templates, as well as clonal amplification due to PCR. In following with our previous cDNA clone naming convention, these new clones have the identifier IP or MIP followed by a 5 number plate well designation (IE: IP01096 =well H12 in plate IP10).