Blasting Primers for RT-PCR - what's hypothetical proteins? My primers always match to these!! (Jan/20/2010 )
I designed some pairs of primers to perform RT-PCR on Taqman machine. Whenever I BLAST any pair, there always appear mRNA of my gene of interest as well as "hypothetical proteins" (to both forward and reverse but interestingly, direction is opposite : plus/plus -> plus/minus and plus/minus -> plus/plus. final PCR product is also reasonable size = ~100bp ).
What are the hypothetical proteins? Are they same as my gene? or different? Then, I can not design any primer pair and do RT-PCR!! I could not find primers really specific to my gene.
What can I do?
Thanks in advance.
it is likely that the hypothetical protein is transcript antisense to your gene and its function is unknown. you need to do a blast search to find out their relationship in the genome. If they are sense/antisense transcripts, try to design primers on sequence that is unique to your gene.
How long are your primers? How good are the other matches to them? Are the other genes to which they match from the same organism? Are the other entries in the not-so-non-redundant database actually just multiple submissions or splice variants of your gene?