PCR optimization

[Austin P. So (Hae-Jin)]

tiredkhan at yahoo.com wrote:

>> High fidelity PCR with an off/on switch mediated by proofreading
>> polymerases combining with phosphorothioate-modified primer.

> Trying to understand what is special about this on/off switch. It's
> already known that you get good specificity with the combination of
> proof-reading polymerase and phosphorothioate 3' end so that mismatches
> can't be edited out. I think this group has shown that the mismatch
> doesn't have to be right at the 3' end but can be up to 8 away.
> 
> Is there something more to it that I've missed? Are there methods in
> which it will be useful to not place the mismatch at the 3' end of the
> primer?

Actually, my intention was not to suggest its "novelty" or whatnot, but 
rather to give it is an example of using proofreading DNAPs with 
phosphorothioate linkages in primers for PCR. I actually think the "wow" 
factor is pretty non-existent, but it has merit nonetheless.

Internal mismatches are less tolerable from a hybridization standpoint 
causing a decrease in the melting temperature, and if it is present 
within the DNAP's binding footprint, it could cause the DNAP to fall off 
or not bind at all (will be DNAP dependent of course). Otherwise, the 
DNAP can still extend off the primer. The phosphorothioate linkage would 
prevent the DNAP from chewing back and editing out the mismatch. The 
terminal mismatch, on the other hand will cause fraying at the mismatch 
site, but because the DNAP can't handle the phosophorothioate linkage, 
it will simply stand idle until it falls off.

I'm not sure which is more advantageous, but since they are applying it 
to PCR, then this "double specificity" is maybe more useful i.e. primer 
specificity and DNAP binding.

Like everything, it probably depends on your application.

Austin

P.S. "tiredkhan" is an interesting handle...