can you judge a 22bp primer based on the 3' 10bp? - (Apr/27/2008 )
To design my primers I usually use primer express and then put them into PUNS to blast them and then do an in silco PCR to check their specificity. Then order them and test them if I get full binding (22/22bp) to the target sequence.
A competitor has recently accused us of using unspecific primers as when they blast the 3' 10bp from each primer pair they get unspecific amplification in silico.
I've tried to recreate this in genome brower http://genome.ucsc.edu/ but they need a minimum of 15bp.
I've also run my products on a 2% gel to double check and only get one band per PCR reaction.
Can anyone advise me as to how, why and if a primer should be judged on only the 10bp at 3' end of the sequence?
The closer to the 3` end you get, the more important the bases become. So the most 3` base (on the very end) is the most important base. Usually if this base is not complementary, the primer will not amplify the target (such as in allele-specific PCR) because this is where the polymerase starts extending the primer. So the last 10 bp are important but it should be looked at as the most important base is the most 3` base, then the second most 3` base and so on, and this should be the focus up to 10 bp if you wish, not necessarily "the last 10 bp" as a unit.
I tried out this PUNS, in silico PCR, can't get it to work, doesn't spit out any results. Know why? I've entered some primers.
what is the difference in the Tm between the whole primer and the 3' 10 bases?
You need to add your primers and blast them against your genome of choice, when the results come back you can then do 'in silico' PCR with them.
This is a good suggestion, what program do you think is best at calculating the Tm as I've heard that different programs give different results?
you can try this one from finnzymes:
quite easy and they claim that the calculation method is very reliable.