'Tandem' Primers for PCR Mutagenesis

Steve Rodems smrodems at students.wisc.edu
Thu Apr 7 23:00:38 EST 1994


In article <940407112930.2831 at jason.uthct.edu>, SHAUN at JASON.UTHCT.EDU
("Shaun D. Black") wrote:

> Howdy All,
>      I was curious if you could use two sets of primers to produce site-
> directed mutants.  The first set are the 'universal' primers that 'cover' 
> the entire length-plus of the gene of interest.  The second set of primers 
> are somewhere within the gene and bear the desired point mutation; these 
> primers would necessarily be complementary to one another.  The 'figure' 
> below illustrates my question:
> 
> 
> Universal,  ________\         ____*__\                  & Mutagenic primers
>            <----------------------------------------    Template DNA +
>             ||||||||||||||||||||||||||||||||||||||||    Base pairs
>            ---------------------------------------->    Template DNA -
> Mutagenic,                   \----*---      \-------    & Universal primers
> 
>                       *********************             Coding region 
> 
> Now run PCR (in presence of dNTP's etc.).  Will Taq 'respect' both primers 
> per strand and amplify a site-directed mutant?  Will significant problems 
> arise from dimerization of the mutant oligos?  Will Taq make a 
> phosphodiester bond when the two growing segments meet?  Any other thoughts 
> or comments would be most appreciated.  Cheers,  Shaun
>   =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 
>   = Shaun D. Black, PhD     | Internet:     shaun at jason.uthct.edu         = 
>   = Dept. of Biochemistry   | University of Texas Health Center, at Tyler = 
>   =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 

I don't know about doing it all in one tube but I've done this same sort of
thing in separate reactions and it works great.  In one tube use one
mutagenic primer and the universal primer with the template to amplify half
of the region.  In another tube use the other mutagenic primer and the
other universal primer with the template to amplify the other half.  Purify
the respective PCR products and then in one tube add both PCR products and
both universal primers.  The PCR products act as the template.  They
overlap where the mutagenic primers are, thus upon denaturing and
reannealing the 3' ends of the PCR products can hybridize and extend to the
end generating a full length product with desired mutations.  From there
the universal primers take over to amplify the full length product.

Reaction A:    ________\
              <---------------------------------------
               |||||||||||||||||||||||||||||||||||||||
              --------------------------------------->
					                         /_______

Reaction B:		                   _______\
              <---------------------------------------
               |||||||||||||||||||||||||||||||||||||||
              --------------------------------------->
                                             /________

Purify products and do Reaction C: (ps: other strand of purified products
won't amplify since their 5' ends will hybridize.)
               _________\
                                <---------------------
                                |||||||
               ----------------------->
                                             /________

I was able to generate random single base mutations over a 9 bp region
using this technique with mutagenic oligos that had a 9 bp degenerate
region in the middle of the oligo.  This method was described in
BioTechniques several months back as well as in the original papers. 
Unfortunately, I don't have those refernces handy.  If you want them e-mail
me and I will find them.
	 
-- 
Steve "Some day I will get the hell out of Wisconsin" Rodems

"Then I am here for the Lee family renioun ... shur-wajo-shur"



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