pcr-primers

Richard Rohan rrohan at world.std.com
Fri Oct 27 07:13:07 EST 1995


"j." <boyson at primate.wisc.edu> wrote:

>But-- just for argument's sake, what if the sequence the PCR primers 
>were designed from was incorrect? or variable? Then, the primers could 
>have a few mismatches. Thus, when the mismatched primer synthesized its 
>strand, that strand will contain "incorrect" sequence, but when the 
>other primer synthesizes its strand, that strand will contain "correct" 
>sequence data, because it's copying the 'real' sequence. 
>
>Could this happen?
>
>
Even if the starting sequence was different from the primer, the excess amount
of primer would "convert" the sequence during each PCR cycle.  The original
sequence would only accumulate geometrically while the primer sequence should
accumulate logarithmically.  If P is the primer and A is the target region and
their respective complementary regions (A' and P'):

         1            2                         3                         4
A'/A + P > A'/A + P/A > A'/A + P/A + P/A + P/P' > A'/A + (3)P/A + (4)P/P' > 

A'/A + (4)P/A + (11)P/P' > et cetera

If the oligo mix is degenerate, it would be possible to have sequence
differences between clones since mismatched oligos will still act as primers.

Also, how close is your oligo region to your sequencing primers?  Depending on
the type of sequencing, read errors can be common in the beginning part of a
sequence (see a recent BioTechniques article).

Rich Rohan
 




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