'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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