Transcription start site determination - (Feb/13/2006 )

I am trying to determine the transcription start site for my gene. I have performed 5' RACE PCR and was wondering if it is a fairly safe assumption that if the 5' end of my sequence obtained from 5' RACE lines up with the 5' end of 3 different EST sequences after a BLAST search, do I have the true end of the 5' UTR?

I tend to think that this gives a pretty good indication but is not confirmation... you could still be looking at a strong stop region in the mRNA (due to secondary structure or high GC content etc) that is reproduced in other ests because it is such a strong stop...

the ways to confirm experimentally are to use the cap-dependent RACE kits (RLM-RACE from ambion) that require that mRNAs be capped (confirming full-length 5' end) or to do high temperature primer extensions (denatures secondary strucutre/strong stops) and get the same sized band... In my opinon, the cap-dependent technique is best because presumably only full length mRNAs can be amplified...may still have problems with strong stops in primer extensions...

HTH

I've used the GeneRacer kit from Invitrogen to map transcription start sites. The kit provides a method for making cDNA only from full-length capped mRNA, so that you don't get false start sites from truncated transcripts.