PCR optimization

Duncan Clark blackhole at abuse.plus.com
Wed Jan 18 10:35:45 EST 2006


Historians believe that in newspost 
<dqk3vn$n1j$1 at nntp.itservices.ubc.ca> on Tue, 17 Jan 2006, "Austin P. So 
(Hae-Jin)" <nobody at nowhere.com> penned the following literary 
masterpiece:
>Michael Sullivan wrote:
>>  On Jan 17, 2006, at 4:53 AM, Duncan Clark wrote:
>>
>>> I would try using a left hand primer that ends with the A mismatch 
>>>at it's 3' end. Preferably use that with a proofreading polymerase 
>>>i.e.  Pfu or a.n.other.
>
>> 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.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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