I am trying to amplify a gene from a cDNA library that I made after mRNA extraction from a fungus. The library itself doesn't seem to be the problem since in my lab we've already easily amplified more than 10 genes and the initial mRNA should be present after extraction as the protein itself is rather constitutively expressed. The thing is that with this particular gene (1900bp aprox.) I could only get the sequence corresponding to one of the domains, the N-terminal region and C-terminal region of the protein (they do not overlap). I was wondering if anyone has any suggestions or ideas for what the reason behind that could be? Could it be cleaved/damaged/incomplete mRNA/cDNA or secondary structure? I tried different sets of oligos, moving around 5' and 3' ends of the gene so I don't think that would be the problem. I also tried to do another batch of cDNA or a cDNA with a gene-specific oligo. I was thinking about buying a synthetic gene with the missing central region and trying an overlapping PCR, but I have no experience in that.
Could you be picking alternatively spliced forms of your mRNA? What happens if you do the PCR with a sense primer binding to the 3' end of the N-terminal domain and an antisense primer binding to the 5' of the C-terminal domain?
Also, I would try amplifying directly from genomic DNA to see what I come up with; perhaps that gene has a central deletion in the strain you are studying.
The protein is not known to have different isoforms, at least not in humans or Yeast, I'm working on a "rare" fungus ortholog but I don't think it would have any additional splicing variant... I haven't tried to amplify the central region with the oligos you mention! Maybe that's something to check!
Yeah, I should try genomic DNA even if there are introns... First I'll have to extract the DNA though... (I only got the RNA by now)!
Thanks for the suggestions!
Perhaps you don't even need to purify genomic DNA. Scrape a bit of the micelium, resuspend it in 200 uL of water, heat to 95*C for 10-20 min. in a thermocycler, and amplify directly from 1-5 uL of the supernatant. Works with yeast, why not give it a try?
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