PCR-RE digest of heterozygotes

Toby Bradshaw toby at stein1.u.washington.edu
Tue Dec 28 14:10:26 EST 1993


In article <CIrCBx.LL9 at umdnj.edu>, Michael Ricketts <ricketts at umdnj.edu> wrote:
>
>If one PCR-amplifies a section of genomic DNA one can cut the product with a 
>restriction enzyme to determine whether such (polymorphic) site is present
> or absent after running the products out on a gel.  However, in heterozygotes
> the amplimers will also form heteroduplexes during PCR which presumably 
>do not cut with the enzyme thus reducing the sensitivity of detecting 
>heterozygotes.  Does anyone have experience of this and advice as how best 
>to overcome this problem?  The enzyme I am using is BsrI, which recognizes
>ACTGG.N and supplier recommends incubations at 65oC.

It's not a problem.  The final synthesis step produces homoduplexes of
each allele.  I do this kind of thing all the time and have never had
a problem like the one you hypothesize.

Toby Bradshaw                       |
Department of Biochemistry          |  Will make genetic linkage maps
and College of Forest Resources     |            for food.
University of Washington, Seattle   |
toby at u.washington.edu               |






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