I'd like to perform RT-PCR to distinguish between transcripts that are about 95% homologous. Does anyone have experience in determining how unique the primers must be in order to detect only the transcript of interest? For instance, if the sequence to which the primer will anneal differs for about 6 out of 20 bases between the 2 homologous transcripts, is that enough? Is there some minimum threshold in terms of the number of unique bases?
I was always told that the last 3 nucleotides at the 3'end are crucial. The end of the primer, where the elongation takes place should be put in the least conserved are, should be there you have your few nucleotides of difference. If there are not three differences in a row, make sure the last nucleotide is different and there are as many possible differences in this are of the oligo. THis way it becomes very unlikely that you get amplification form a highly similar gene. If you do this for both primers you should be save since the polymerase with have trouble to work with the nonannealed bases at the start of the elongation.
I agree with Mr. Andl, and I would like to add one from my experience, 6 out of 20 mer is enough being distinguishable ONLY IF one of the base is on 3'. The rest is depending on how high you could raise the RT temperature.
Thanks for the feedback. I did manage to find 2 regions each 6 bases long that only have 1 or 2 bases in common between the 2 transcripts. I will design my primers such that the 3' end includes these regions. I have heard that a 3' "clamp" is really important. Justin, thanks for the suggestion about using the software. I will give it a try.
Hi, I agree with everybody in the point of having the different nucleotides on the 3' of your primers and that the last nucleotide is the most important to avoid cross amplification.
I wanted to add that you can check if you are amplifying only the desired transcript by sequencing the PCR product.
good luck!
- Badges
- Science method