PCR-RE digest of heterozygotes
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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