'Tandem' Primers for PCR Mutagenesis

Bernard Heymann bheymann at bragg.bio.purdue.edu
Fri Apr 8 12:44:59 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've tried it! In my case it didn't work, which doesn't mean it will never
work. I got the three expected fragment sizes, but no mutagenenic product.
Perhaps the severe dilution of the nucleotides by synthesis of undesirable
products makes this a low-efficiency method.

-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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