I want to clone cDNAs containing especially the 5’-most sequence of some genes-of-interest that are relatively large and whose 5’ region is not associated with any WormBase-curated ESTs (e.g., yk clones). I’m also on a tight budget. So I was wondering if it were possible to isolate 5’ cDNA sequences in an RT-PCR reaction using – in the PCR step – forward primers corresponding to a C. elegans splice leader sequence (e.g., SL1) in combination with reverse primers corresponding to known downstream sequence. See any problems with this approach? I was hoping that perhaps it might be a viable and frugal alternative to using conventional RACE.
RT-PCR with SL1 (or SL2) primers been used successfully for some genes, and is probably always the first thing you should try, because it is, as you say, rapid and cheap. What I don’t know is what proportion of genes apparently aren’t trans-spliced; I worked on one for which I could never find evidence of trans-splicing, after trying pretty hard, so I’m pretty sure they exist, but I never found anything about their prevalence. In such a circumstance, I preferred RNA ligase-mediated RACE to standard 5’ RACE; it gave more consistent results.
That is reassuring – now I can proceed with confidence. I’m fairly sure that at least some of the genes-of-interest are trans-spliced. There are multiple tracks available for viewing trans-splicing data in the genome browser, the most recent being from modENCODE (Waterston group, I think). The modENCODE tracks indicate whether SL1 or SL2 trans-splicing was detected by deep sequencing under various conditions or at various developmental stages.
Thank you for responding to my question and providing additional insight.
I have had very good luck using the “FirstChoice RLM RACE kit” from Ambion (now bought out by one of the big companies). This is the method mentioned above by Hillel, but I wanted to mention this specific kit. You end up with clones of the 5’ end of your gene that you can sequence to make sure they all start at the same base. Sometimes I have found differences of 1-3 bases in transcription start site between clones, but you will get the beginning of the protein for sure.
-Tim Kroft