making primers for similar protein sequences - making primers for similar protein sequences (May/29/2006 )
Hello all,
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!
Thanks
Take care
-Nawaaz
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?