PCR Design

Aawara Chowdhury via methods%40net.bio.net (by aawara from FEMA-trailer.org)
Wed Oct 11 06:28:15 EST 2006


In <5MpGZODTuKLFFAgx from abuse.plus.com>,
 Duncan Clark <blackhole from abuse.plus.com> wrote:

> Historians believe that in newspost 
><diLWg.22242$vi3.640 from bignews3.bellsouth.net> on Tue, 10 Oct 2006, Aawara 
> Chowdhury <aawara from FEMA-trailer.org> penned the following literary 
> masterpiece:
>>> Speaking of which: What is expected % of PCR products with added Ts
>>> overgangs when using Taq? The question recently poped up discussing
>>> a particular experiment and I relized I have no idea. So, what is the
>>> frequency? 10%? 50%? Nearly 100%?
>>>
>>
>>The propensity of Taq to create products with a single base non-templated
>>3' dT overhang is nil.  But close to 100% of the products will have a single
>>base non-templated 3' dA overhang.
>
> But is also very dependant upon the 5' sequence of the primer according 
> to an 'old' genotyping Biotechniques paper. By altering the primer 
> sequence they could ensure that 100% of products had the overhang and 
> defined a the required sequence to achieve this. Not sure of the exact 
> reference though :-(

That's correct.  My recollection is that if the 5' end of the primer
begins with a "dG"; i.e. the last templated 3' nucleotide is a "dC",
then the propensity to add an additional non-templated "dA" is very
close to 100%.

AC
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