site-dir mutagenesis to insert RE sites

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Oct 29 15:09:14 EST 1992


<>Dear PCRologists and homebrewers,
<>I need to do some mutagenesis on a 500 bp stretch of DNA, to
<>create restriction sites about every 100 bp. I haven't done
<>any multiple site-directed mutagenesis since the dark ages B.P.
<>(Before PCR) and haven't kept track of the methodology papers.
<>For cost reasons, I would prefer to make only one mutagenic
<>primer per site. Has anyone out there tried (with success) the
<>"megaprimer" method (Sarkar & Sommer BioTechniques (1990)
8:404-7)?
<>Or do you know a better method?
<We have used the "megaprimer" approach with great success... in
fact we
<*thought* we had invented it and then found the reference cited
above after
<the fact.
<I still worry about umwanted mutations creeping in through PCR,
and
<would suggest subcloning back a small piece that you sequence in
its
<entirety.
<dennis

I have another suggestion.  Only difference, need two oligos per
site.  I have had good success with this method in introducing 2
sites in a 3.5 kb fragment.  Also, the "mega primer" method did not
work for me, I think because of the size.  In this method oligos
containing the RE sites by silent mutagenesis are synthesized to
encompass each RE site.  Separate PCR with each set of oligos will
yield you "n+1" fragments for each "n" sites desired to be
introduced.  Now you can digest the fragments with appropriate RE,
purify the frags and ligate in a single tube.  To ensure formation
of the full length fragment in right orientation, you can choose RE
sites that yield unique overhangs.  The final fragment can be
eluted from a gel and cloned back.  Also, we have published methods
and a computer program which will help you identify a large number
of RE sites that can be introduced by silent mutagenesis (was very
important to me).

A crude illustration is

========           ===========          ========
        ===========           ==========         =======
                           |
   RE digest, ligate,      |    and clone it back

The easy use of this method requires that you select unique
RE overhangs for each site.
The relevant references are
Shankarappa et al BioTechniques 12: 382-384
Shankarappa et al BioTechniques 12: 882-884
Shankarappa et al PCR Method Applic. 1: 277-278

The computer program called SILMUT identifies amino acid motifs
that are suitable for the introduction of RE sites by silent 
mutagenesis.  The TABLE program generates a table of amino acid
motifs that are obtained by translating the RE recog sequence in
three reading frames.  We are making the SILMUT and TABLE programs
available for free by anonymous ftp from phobos.med.pitt.edu. in
the directory pub/bsh.  You will have to use the binary option to
download the ".exe" files.  The README file is kind of outdated, so
please send me a mail I will send you a clearer README file.  Very
shortly, I will be making these program available on bioftp at
Indiana and Quantum Chemistry Program Exchange at Indiana.  I will
also be very happy to provide the program to anyone if you can send
me a blank disk and a mailer.
Raj Shankarappa
bsh at med.pitt.edu
Dept of Pathology
736 Scaife Hall
Univ of Pittsburgh School of Medicine
Pittsburgh PA 15261
(412) 648-9763
 



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