Site-directed mutagenesis

Robert M. Horton, Ph.D. rmhorton at ATTOTRON.COM
Fri Sep 19 15:18:11 EST 1997


<a.doherty at bris.ac.uk> wrote:

> medbh at biovax.leeds.ac.uk wrote:
> >
> > Could anyone recomend a PCR based method of site-directed
> mutagenesis for a cDNA clone for cell expression studies, that has
> successfully worked well, as there are so many on the market to choose
> from.
>
> YOu don't need a kit - if the site you want to mutate is near a unique
>
> restriction site, amplify from that site and incorporate your mutation
>
> in the primer. THe ends of the PCR product can then be snipped off, or
>
> the product can be cloned into a TA vector (we use pCR2.1 from
> Invitrogen). If your mutation site does not lie near a unique
> restriction site (most likely), amplify two products which overlap the
>
> mutation site. Introduce the mutation in the reverse primer for the 5'
>
> fragment, and in the forward primer of the 3' fragment. Purify the two
>
> fragments, and use them as a template for a second PCR using only the
> non-mutagenic primers for those fragments.This should give you a
> fragment going from the 5'end of the 5' fragment to the 3' end of the
> 3'
> fragment, effectively joining the two PCR products together. Ensure
> that
> the ends of the final fragment has unique restriction sites, and clone
>
> either into your coding sequence or a TA vector. Then sequence the
> final
> product to ensure the mutation is there. We've used this method
> successfully for both mutagenesis and epitope tagging.

Some references for PCR-based directed mutagenesis by overlap extension:

Horton RM, Ho SN, Pullen JK, Hunt HD, Cai Z, Pease LR. Gene splicing by
overlap extension. Methods Enzymol 1993;217:270-279

Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed
mutagenesis by overlap extension using the
polymerase chain reaction.Gene 1989 Apr 15;77(1):51-59

--
              Robert M. Horton, Ph.D.
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