Hi all, I hope someone can help me or at least give me an idea where I can go from here because I have run out of ideas. I've been looking for human orthologues in the zebrafish genome and have identified 3 of them. One of them looks perfect, with a very good synteny match and the amino acid identity between the zebrafish and human is very high (~70%). So I made primers and amplified up the transcript and managed to locate from exons 6-14 (the human transcript has 15 exons). Following this did a 5' RACE to try and locate the very beginning of the transcript and have hit a dead end. When I put the sequence through an ORF finder, I get an extra exon (let's call it exon 5 for now) and everything else before that pretty much gives me stop codons. Assuming that this is a shorter transcript then what I thought it'd be, it doesn't start with a methionine. Is that possible?
Should I redesign some primers and do another 5' RACE and hope I get something? Or is there a way where I can identify splice sites from the raw sequence and work from there?
Edited by moerae, 23 November 2009 - 07:37 PM.