Dear all,
I am trying to design primers for a AS-PCR and I am facing some problems with one of my target SNPs. As for the others, I have been considering the following when designing the AS primer: The target SNP [C/G/T] will be the base at the 3’end of the forward primer. So I have designed three forward primers and one common reverse primer. When PCR is performed with these three sets of primers PCR products of desired size were amplified from the same sample.
Anyone could help me for the best solution for this problem? any suggestions ?
you can add dmso in increasing amounts until only the one primer can anneal or increase the annealing temperature in a gradient to see what temperature each forward primer anneals at without the other primers annealing, This will need samples of known sequence to sort out which primers work when others do not, Your results looklike 2 primers can anneal over one base change so this needs to be sorted. Alternatively you can design dcaps primers where you introduce a second change to the sequence in the forward primer and depending on the original sequence it creates a restriction enzyme cut site which identifies exactly what the original sequence was, Google dcaps and it takes you to a site that designs the primers for you,
With AS pcr sometimes moving the primer so that the non annealing base is second or third in from the 3' end works better than having the match/mismatch exactly at the end of the primer
Yes I agree, someone suggested this for me in the past and it worked.
I have learnt many things from the comprehensive views of Prof. Paul. Thanks
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