graft pcr, help needed********

Basavaraju Shankarappa bsh at MED.PITT.EDU
Wed Sep 16 10:35:27 EST 1992


> Rod Bonfiglioli (rbonfigl at waite.adelaide.edu.au) wrote:
> : does anyone please have some information or protocols on "recombinant" or
> : "graft" pcr....
> Rod-
> 	Here are some references you might want to look at:
> Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R.
> "Engineering hybrid genes without the use of restriction enzymes: 
> gene splicing by overlap extension".  Gene 77:61-68, 1989.
> Horton, R.M., Cai, Z., Ho, S.N., and Pease, L.R. 
> "Gene splicing by overlap extension: tailor-made genes using the 
> polymerase chain reaction". BioTechniques 8(5): 528-535, 1990.
> Horton, R.M., and Pease, L.R. "Recombination and mutagenesis of DNA 
> sequences using PCR". In: Directed Mutagenesis: A Practical Approach. 
> (ed. M.J. McPherson) pp 217-247, IRL press, 1991.
> Sarkar, G., and Sommer, S.S. BioTechniques (1990) 8:404.
> Disclaimer:"of _course_ I'm tooting my own horn!" 8)
> --
> Bob Horton            /\ "Crash programs fail because of the theory that
> U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
> 1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
> St. Paul, MN 55108    ^^   horton at molbio.cbs.umn.edu/(612) 624-3790

One small comment arising as a result of experience.  It is perfectly fine
to use any of the above and other methods if you are manipulating DNA
of few hundred basepairs.  I have had terrible results when trying to
use similar strategy when the fragments I was trying to manipulate was
several kb.  I got big smears instead of clean fragments.

One possible alternative to overcome these problems is to introduce unique
restriction enzyme sites adjacent to the region targeted for mutagenesis.
This method provides a easy handle to manipulate the small fragment by any
of the available methods.  It is quite easy to identify unique RE sites that
can be introduced by SILENT MUTAGENESIS.  If you take the RE site and translate
it in three reading frames, you generate a huge set of peptides that will be
compatible for the introduction of that RE site.  Once these have been identi
fied, these sites can be introduced by amplifying using the mutant primers,
digesting to create unique overhangs, ligation followed by ligation back into
the parent vector.  Now you can cut out the fragment targeted for mutagenesis
and insert any sequence back.  
Although the explanation looks lengthy, it is quite simple and easy to 
accomplish.  May be you will not get results in one day, but your chances of 
getting the right result is very high.  
To toot some of my horn, following are the relevant papers that may be useful.
Shankarappa et al., BioTechniques 12:382-384
Shankarappa et al., BioTechniques 12:882-884
Shankarappa et al., PCR methods and applications 1:277-278.
Raj Shankarappa
Univ of Pittsburgh
   
 



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