'Tandem' Primers for PCR Mutagenesis

GEORGE XIAOXI OUYANG, ELECTRICAL ENGINEERING, PHONE 579-0970 ldeng at kean.ucs.mun.ca
Sun Apr 10 11:42:34 EST 1994



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

Hi! If you do two seperate  PCR each with one universal and one mutagenic 
primer and manage to get the  two bands  of interest. Then purify the bands 
from  the agarose gel with Qiagen or Geneclean, add DNA from each recovered 
band to Another set of PCR with the two universal primers. If the condition 
of PCR is adjusted o.k. You won't have problem to get the mutated product. 
Good Luck!  Langtuo Deng



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