'Tandem' Primers for PCR Mutagenesis

Shaun D. Black SHAUN at JASON.UTHCT.EDU
Thu Apr 7 11:29:24 EST 1994


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 = 
  =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= 



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