Non specific amplification - How to check if the primers give non specific amplification or not (Jan/19/2007 )
Is there any software or a website where I can check that if my primers give some non specific amplification on a particular sequence.
I used the aligning techniques to check the binding of the primers. The problem is I have two similar template sequences (but they are different). I have designed primers for one of the sequence, now I want to check that these primers may or not give a non specific amplification of the other template sequence.
My primers dont fully bind to the second sequence but give a 88% similarity to the non desired DNA sequence.
Thanks for any help.
Check with BLAST (NIH wesite), it will tell you if there is another area in the genome that have similarity with it and also if there is another specie with the same sequence.
What software do you use to design your primers?
I use the software at IDTDNA to design and order my primers. You can blast from right within the software.
You have to register for an account, but it is free.
I always use primer3, easy and free!!!!
I have two pieces of advice.
Focus on the 3` end of each primer and make the last 10 bases or so as dissimiliar as possible. This region of the primer is going to be most important in specific isolation of your target.
It might be a good idea to design some nested primers (i.e. primers that will anneal to the outside of your sequence of interest). Because you're trying to isolate a particular target you probably don't have much room for playing around with the primer sequence right on the ends of that target. You may be able to find some more dissimilar sequences outside your sequence of interest and once you have isolated that target you can use that template and your original primers to amplify the sequence you require.