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Taq sequencing

Phillip San Miguel pmiguel at purdue.edu
Fri May 7 04:08:14 EST 1999


"Mary P. Remington" wrote:

> I am curious at to whether Taq sequencing can introduce base pair changes
> in a sequence like the Taq polymerase can in Taq polymerase PCR.  Is
> anyone familiar with that type of problem and what could contribute to
> this?  Thank, Mary

    No, using Taq polymerase for sequencing will not introduce errors into
your sequence.  More precisely, it will introduce errors at a rate so low, you
will not be able to detect them.  That is, oh, say 1 in 1000 molecules, at any
one position on your sequencing gel will be wrong, but the 999 correct ones
will swamp out the "noise" of the incorrect one.  (Also, Taq polymerase is
unlikely to make even that many errors.)
    PCR does exponential amplification because it utilizes two primers, while
sequencing reactions using a thermostable enzyme and one primer does a linear
amplification.  That is, during PCR each copy of a target sequence could
(theoretically) undergo a doubling each cycle.  This is because each product
produced during a reaction cycle can be a template for a new product the next
cycle.  But in a sequencing reaction, there is only one primer extended.  This
product *cannot* be used as template the next reaction cycle.  Thus with
PCR:20 cycles, 2^20=1000000-fold amplification of the target; with sequencing:
20 cycles, 20-fold amplification.  If an error occurs in a fairly early step
in PCR, it will tend to be amplified to the point that it might be cloned.

Phillip San Miguel




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