Identifying a new protein isoform

[talkingtree]

Hi,

I'm trying to obtain a new DNA sequence of an unidentified protein isoform. I suspect it is an isoform of a known protein. So far I had generated the cDNA and was able to parts of c terminus. The c terminus could be a common domain for all isoform. The N-terminal domain could not be amplified using the known DNA sequence.

Illustration below demonstrates what I think. The top 2 dots shows the primers that work for the at the c terminus. The second line is the DNA sequence I want to amplify (especially towards the N terminus)


- -
5' ------------------------------------------------------------------ 3'

Is there a new/quick method of obtaining the whole DNA sequence?

[mchlbrmn]

If you try the "Code" button at top when entering your reply, and paste something from a text editor, or type between the two bracketed words, you can get your little diagram in your reply to come out next time.
I think what you want is RACE, Rapid Amplification of CDNA Ends. This lets you PCR from a known sequence to the ends of the RNA transcript. There are kits sold to amplify to the poly A, and/or the 5' capped end.
Did you already check Genbank and the EST and RNA transcript libraries? If you have a protein sequence, you can do a protein to nucleotide BLAST search, tblastn, I think it's called.

[relaxin]

You can get the 5'-sequence using a 5'-RACE kit. I used the kit from Clontech before, it worked fine on the first try.

I think before you buy the 5'-RACE kit, it may be wise to put your sequence on blastn search to see if the full-length sequence has been reported:

http://blast.ncbi.nlm.nih.gov/Blast.cgi ... =blasthome