Restriction Site PCR
RBHR at BPHVAX.BIOPHYSICS.ROCHESTER.EDU
Fri Apr 14 22:57:15 EST 1995
From: IN%"fordb at BCC.ORST.EDU" "Bryan Ford" 13-APR-1995 23:58:25.38
To: IN%"RBHR at BPHVAX.biophysics.rochester.edu"
Subj: Restriction site pcr
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Date: Thu, 13 Apr 1995 20:54:30 -0800 (PST)
From: Bryan Ford <fordb at BCC.ORST.EDU>
Subject: Restriction site pcr
To: RBHR at BPHVAX.biophysics.rochester.edu
Message-id: <75271.fordb at bcc.orst.edu>
Hello Dr. Harrison:
I was just reading the news as is my custom about once a week and noticed
your inquiry about restriction site PCR. Shortly after the publication of
their article in PCR Methods and Applications, 2:318, 1993, I began to
consider the use of this method to "walk out" into unknown intronic
sequence from well characterized cDNA sequence. Eventually my curiosity
became great enough to phone Dr. Sarkar at Mayo. He very kindly answered
my questions and I will try to relay the essence of his answers from the
brief jottings I made at the time. My first question was "Do you
restriction digest the template as a first step in restriction site PCR?"
The answer was a definite "No", (the restriction sites apparently serve
only as a means of defining a location, at least statistically, for the
annealing of the RSO. My second question mirrors your own: "How do you
generate the arbitrary spacer sequence?" The anwer was "It is not
arbitrary but it is truly random." That is, he assured me they simply
allow the oligo synthesizer to use a mix of the dNTPs for the 10 or so Ns
in the "spacer". I then asked him what brands of thermostable polymerase
they had successfully used. The answer was "We have only used Amplitaq."
Finally, I asked him if he used "cold-start", "hot-start" or room
temperature setups. He gave a technical reason why they use room
temperature setups for this method, which I recall related to the need to
encourage some non-specific annealing across the NNNNNNNNNNN spacer.
Other researchers in my building actively pursued "restriction site PCR."
I recall that they changed the PCR parameters to add of a couple of
initial cycles at a quite low annealing temperature (37 degrees or
thereabouts) before proceding with the rest of the PCR at annealings more
suited to the primer length. This brought up some reproducable products.
I have not spoken with these researchers about this work lately, but my
impression has been that they have now abandoned the method and they
have returned to inverse PCR, which, although tedious, has worked well.
If you are still seeking means of easily "walking" from known sequence I
would appreciate a line or two from you as to the specifics of the
situation since I have tried a number of approaches to this problem and
would like to talk shop with others who may be looking for the best ways
to do it. I can certainly describe some very plausible schemes that did
not work, in our hands at least.
I hope this discussion is useful to you. I am unable to post to the
news under my current server. If you wish to post this email to "methods
and reagents" I would have no objection. It might benefit others.
Bryan L. Ford
Marine/Freshwater Biomedical Center
Oregon State University
Corvallis, Oregon 97331-6602
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