Problems with the Specificity of the Primers
Posted 27 October 2010 - 10:12 PM
I designed TaqMan assays for various sequences. However I found that in some cases, my reverse primer was specific and did not bind to any other sequences on BLAST search and forward primer was entirely homologous. Will such a primer pair work? Also if 15/16 sequence bases are homlogous to other sequences, should I still consider it to be specific since 1 base should be enough to make my primer unique to amplify?
Posted 29 October 2010 - 08:44 AM
It depends on the position of the mismatch base, mismatch on the 5' end or in the middle doesn't matter at all, and usualy even 1 bp mismatch on the very 3' end isn't enough to stop nonspecific amplification as many alele-specific PCRs use 2 bp mismatch on the 3' to differ between two SNPs. So that primer is not specific.
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Posted 01 October 2011 - 06:28 PM