splicing genes with pcr

Bob DeLisio delisior at rnisd0.DNET.roche.com
Wed Apr 21 11:31:31 EST 1993


jurgenson at iscsvax.uni.edu writes:

>I have just amplified portions of two genes which I wish to splice together
>with pcr.  The primers used to amplify the fragments should have produced 18
>basepairs of overlap between the 3'end of one and the 5' end of another.  We
>have tried to splice these fragments together with pcr using the outside
>primers but get only a smear from high to low molecular weight.  Does anyone
>know the solution to getting this to work?  please post as this may be of
>general interest. thanks

We make constructs frequently this way and have found that it works with
higher efficiency if the primers are designed such that there is a T in the
template following the calculated end of the PCR product. This method takes
into account the fact that Taq pol adds non-template directed bases to the
"finished" product. Fortunately, the polymerase exhibits a strong preference
for adding an A in this position which makes this technique work.

Regards
Bob DeLisio



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