argh more pcr headaches! - (Mar/11/2010 )

Can this scenario be possible:

the 3' end of the primer does not anneal to template strand and instead hangs away from the template dna while the 5' of primer remains attached. Could Taq pol make a new strand in this scenario???? the person who supervises me seems to think so. i on other hand cant see how it is possible as taq can only bind to 3' end of primer and extend it the primer. i dont know, this person has years more experience than me but i feel in this instance he is wrong. would appreciate the help
Danke!

In my experience a single base mismatch at the 3' end is fatal for extension, and I believe is widely used as a means of identifying SNPs easily.

phage434 on Mar 11 2010, 10:20 AM said:

Hi Wiz,
Just curious, I'm no primer designing pro, but working on it, how did you determine that the 3' end of your primer isn't annealing?

Well, you have a primer which differs from the template sequence in a single position at the 3' end. If you are detecting SNPs, then two primers, one of which matches each possible base in that position can be run, and whichever one amplifies identifies the base at the 3' position.

phage434 on Mar 11 2010, 07:53 PM said:

Phage,
I'm confused. Maybe I'm not understanding what wiz is trying to do but my first question:
If you run a normal PCR rx to amplify a region of interest and it fails, how do you determine that one of the primers isn't annealing on it's 3' end?

Second, I understand somewhat your explanation of detecting SNP's.
Question is: If you need an exact match on the 3' end of a primer to get extension, wouldn't you need four sets of primers (ex. four different forward primers with the same reverse primer), and the only difference between them to be a different base in the suspect position?

I presume you know the sequence of your template. If not, you should sequence it, or use a primer to a section that has good sequence that you know.

Usually common SNPs are replacement of a specific base by another specific base. If you truly did not know the replacement base, then yes, you'd need four.
It's an expensive way to sequence a single base.

phage434 on Mar 11 2010, 10:35 PM said:

ah hah............this sounds like what one might do to verify an SNP after getting back sequence that shows a heterogeneous mixture when looking at the chromatogram.
got it..... thanx phage