making primers for similar protein sequences - making primers for similar protein sequences (May/29/2006 )
I'm working on making pcr primers for 5 ephrin A (A1-A5) rat proteins. These proteins are very similar, although two of the proteins (A1 & A3) are about twice as long as the other three(mRNA is twice as long). These primers are to make probes, about 200-300 bp's long, to be used later on for in situ hybridization. Here are some of the problems I'm facing with this so far:
- I can't make the primers for A1 & A3 outside the region of aligment with the other three, since there is a chance that the other three are not all complete sequences, which will cause faulty hybridization of other proteins.
-These five proteins are very similar, and when aligned, there are many regions of homology, and none long enough to give me a 200-300 bp region in between forward and reverse primers.
Here are some questions I was wondering about:
- if I'm making a primer of about 10-20 bp's long, what should be the smallest amount of homology between one primer and another for a different protein ( I was told that about 10-15 bp similarity will still help keep specificity, but I'm not sure).
-I'm trying to find a program or database that will help me find a conserved domain or region in all five proteins, but that can also tell me this region in the mRNA code, so that I can align these proteins again, identify the regions of homology in each, and then look outside these regions to try and find my specific primer.
I hope that made sense...
any help on this topic would be very much appreciated!
You need to be doing this at the DNA level. Similarity or identity at the protein level means nothing when designing primers to produce a DNA probe. Are the DNA sequences of these genes available?
Yes I have the full DNA sequences for all of these proteins.
Okay, so to make a primer set that will amplify one of the genes and not the others, you need to align the DNA sequences, and find a DNA region that is unique for your gene -- especially with regard to the 3' end of the primer(s).
I've aligned the DNA sequences , and identified the regions. I used primer 3 to make my primers, and now what I'm doing is blasting each left and right primer for each protein to see if they match with anything else. I will also be blasting the regiong between my primers, since the regions in between will be my riboprobes, and I dont' want them hybridizing with other parts...
How does that sound?