what affects primer specificity? - (Aug/14/2007 )
Hi,
Can anyone give me some ideas about what might affect primer specifictity? How different does the primer have to be from the template sequence until it won't amplify it?
I've designed some specific primers, but it continues to amplify sequences I dont want, that differ in about 3-4 bp. Ive increased the annealing temp (its now >70 degrees!) to no avail.
Sounds like there you have been unlucky and used a primer that closely matches several sequences in the genome. There is no way around this other than to redesign the primers. To increase the primer specificity you could make them longer, this would reduce the chances of having a closely matching sequence elsewhere in the genome. Also, if your organism has genomic sequence available, you could try a blast search to see where the primer is annealing.
Can anyone give me some ideas about what might affect primer specifictity? How different does the primer have to be from the template sequence until it won't amplify it?
I've designed some specific primers, but it continues to amplify sequences I dont want, that differ in about 3-4 bp. Ive increased the annealing temp (its now >70 degrees!) to no avail.
At the most basic, if the 3' base is wrong, the primer won't extend. Next, you have the question of internal mismatches. The more mismatches, the lower the temperature that the primer will fall off. (Remember that the Tm is the temp at which 50% of the primers bind to available sites.)
As bitesize... suggested, I'd consider BLASTing the gene to find where the primers are (mis)priming.
If you can't change the primer sequence, you could consider additives like DMSO or betaine, which destabilize duplexes. That might allow you to get just your desired product.
You can also try a touchdown PCR, where you start well above the optimal Tm, and drop the annealing temp 1 degree every 2 cycles. You won't know the optimal annealing temp, but if your'e only after a specific product, you might not need to know.