PCR optimization

[Austin P. So (Hae-Jin)]

Duncan Clark wrote:

>>> I would think a non-proofreading polymerase should be used in this 
>>> case. Otherwise, the polymerase might remove the 3' terminal primer A 
>>> prior to extension. Unless my reasoning here is wrong. In any case, 
>>> iI agree that the best way to discriminate between the two plasmids 
>>> is by having the mismatch at the terminal nucleotide of the primer.
> 
> 
> True. I was thinking about stopping the enzyme extending in the first 
> place with a mismatched base but then of course with Pfu, if it does put 
> the wrong base in the proofreading exo will remove it. I was trying to 
> be too clever :-(
> 
> Papers using single nucleotide extension assays or 3' discrmination 
> primers for SNPs may be the place to look for pointers.
> 
>>
>> People have introduced 3'-terminal phosphorothioate linkages into the 
>> primer which would prevent editing of the 3'-end by the proof-reading 
>> polymerase (I think it was with Vent, although in principle it should 
>> work just as well with Pfu).
> 
> 
> An NAR paper many moons ago but widely used for the phi29 polymerase 
> rolling circle amplification methods which use hexamers with 
> phopshorothioate linkages. Standard hexamers get chewed up.

Here is one (I think the other one was by Di Guisto...?)

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

Yang HL, Jiang HJ, Fang WY, Xu YY, Liao DF, He FC.

SNP Institute, Institute of Pharmacy and Pharmacology, Nanhua 
University, Hengyang 421001, Hunan, China.

In the initial report, introducing a single phosphorothioate 
modification at the very 3' terminus of the oligodeoxynucleotide primer 
has been shown to effectively protect the oligodeoxynucleotide 
degradation due to the 3' exonuclease activity. In this study, we 
reported a novel finding that phosphorothioate modification at the 3' 
end of primers could not only effectively prevent the primer from 
degradation, but could also mediate an off-switch extension by Pfu 
polymerase when primers also carry single or multiple mismatched bases 
located in the first eight bases of the 3' terminus. This suggests that 
the combination of 3' phosphorothioate-modified primers with exo+ 
polymerases such as Pfu constituted an on/off switch, which allows 
perfectly matched primers to be extended but not mismatched primers. 
Furthermore, we found that polymerases with different fidelities showed 
different efficiencies in turning off mismatched-primer mediated 
extension. So we described here a SYBR green-based real-time 
quantitative PCR assay for the detection of abundance level of gene 
expression that did not require fluorescently labeled gene-specific 
probes or complicated primer combinations. The emergence of real-time 
quantitative RT-PCR technology is thus suited for a diverse application 
with a need for high-throughput methods to detect and quantify different 
gene expressions by way of simplicity, versatility, and accuracy, and 
thus could complement global microarray-based expression profiling 
strategies.

Cheers

Austin