I am trying to design primers for a tomato virus isolate and find that I need to allow two consecutive ambiguous nucleotides for the last two bases at the 3' end.
Hi, Rudolph. It does not seem to me to be a good idea in terms of specificity. You can use Inosine or 5-nitroindole (universal bases with different degrees of pairing preferences) or other modifications available elsewhere. Why cannot you ignore this ambiguous site?
I agree with Peter. If you do not have proper base pairing at the 3' end of a primer, you will not get efficient extension (or any extension) by the polymerase. As Peter suggested, inosine or 5-nitroindole can take care of the base pairing, but it would be better to eliminate the ambiguous site and extend the primer at the 5' end.
Why do you want this at the 3' end and not the 5' end. It does not matter what the length of un-pairing at the 5' end , it will not affect the PCR amplification. But even single base pair at the 3' end will affect. Are you putting exactly at the 3' end or near to the 3' end ?
Not a good idea to allow mismatch at the 3' end as any amplification is very unlikely. You can have the ambiguous nucleotides but extend the primer so that those two nucleotides are at the center or 5' end and not exactly at the 3' end.
So the answer is NO, you cannot have two consecutive ambiguous nucleotides for the LAST TWO BASES at the 3' end.
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