From: wetsel_r@wums.wustl.edu
Newsgroups: bionet.molbio.methds-reagnts
Subject: Long PCR's - revisted
Date: 24 JUL 92 12:20:22 GMT
Organization: MIT PLASMA FUSION CENTER
NNTP-Posting-Host: msnews.wustl.edu

Dear Netters:

Please forgive the disorganization...  through discussions of PCRs and 
Taq-like enzymes, I've interacted with a number of people and I've ammassed 
replies & messages together.  Somewhere below, I hope there is something of 
use to you...

The initial posting (which I have since misplaced) mentioned that a lab had 
used USB's Delta-Taq for PCR even though it isn't advertised for use in 
PCR.  The add is geared more toward sequencing and due to legal manure, USB 
won't come out and tout Delta-Taq for PCR.  

I hope you don't mind the length of this message but we will get to long PCR's
shortly...

What follows are edited answers to questions that have been posted on the 
bionet.molbio. sub.
----

The delta Taq was actually developed here at Washington University by Dr.
Wanyne Barnes.  His common name for it is Klen-Taq and he exclusively uses
it for PCR.  He'll often use upwards of 50U in a reaction for long PCRs and
obtain lengths of 15-17kb!!!  I personally have obtained 5 kb PCR fragments
using the enzyme (and HotTub too!).  Because of patent rights by Cetus, USB
doesn't want to deal with the legal hassles of adveritsing it for PCR, but
in fact, that's what Wayne designed it for.  What do I mean by designed? 
Wayne whacked off the 5' end (235-aa) of the polymerase so that Klen-Taq
(Delta-Taq) has virtually no exonuclease activity.  It and Hot Tub are the
best PCR enzymes we've ever used in our lab. According to Wayne and his
tests, Vent polymerase actually has an unacceptably high exonuclease
activity such that one can even lose restriction sites that were designed
in your oligos. 

----- 
>Do you have a protocol for getting long PCR products?  And who makes Hot
>Tub?  (We have seen ads for it, but can't recall right now who or where.)  

Yes.  I'd like to think I can help.  I'd suggest that you also get the 
referece Anal. Biochem. 202:46-49 #46, entitled In Vitro Amplification of 
DNA Fragments > 10 kb.  By Kainz et al.  These authors used HotTub and
obtained PCR products as the title says, over 10 kb.

Hot tub is made by Amersham and we have had MUCH better results with it 
than Ampli-Taq.  [Disclaimer: we are not employed nor do we represent Amersham
or USB - we just use some of thier products...]

Wayne Barnes and Delta-Taq:  First, I should state for the record that W_B 
showed an experiment to me where he determined Taq activity.  He conducted 
a PCR reaction and after phenol, clfm, phenol-clfm, 100% and 70% EtOH 
steps, he removed a small amount from the aqueous phase.  To this he added 
32P-dCTP and incubated this small volume at 37'C. Incorporated dCTP was
separated from free label, and the material spotted onto a membrane and
exposed to X-ray film.  At each step, Taq and Delta-Taq demonstrated polymer-
ization activity.  In short, the enzyme retains activity through phenol, clfm,
phenol-clfm, 100% and 70% EtOH procedures.  He feels that given the activity,
the enzyme can end-fill after restriction digestion.  The only treatment which
shut down Taq activity was precipitation using PEG - the mechanism of PEG
inactivation isn't yet known. Thus, before W_B digests, he PPTS with PEG.
Specifically, he freezes the PCR amplification tube at -20'C and removes the
mineral oil and washes twice with 5'C clfm.  After thawing, he adds 10ug of
glycogen and 1/2 volume of 30%PEG-3350/1.5M NaCl and lets it stand for 30 min
at 4'C.  CFG for 8-10 mins in microfuge, wash with 70-75% EtOH and dries it. 
This only works if your PCR fragment is over about 500bp.   If he didn't do
this he (and our lab for that matter) has had some difficulty in subsequent
ligation steps that W_B feels is due to residual Taq end-fill activity.  His
studies with Delta Taq have been published in "GENE", Vol 112, #1,pg 29-35,
1992.  The article is entitled "The fidelity of Taq polymerase catalyzing PCR
is improved by an N-terminal deletion".  This is where he describes KlenTaq
which is marketed as "Delta-Taq" by U.S.B..

W_B in his paper used oligos that were 40-45mers.   W_B uses a slightly
different buffer: 20mMTrisHCl pH 8.5, 2.5mM MgCl, 16 mM(NH4)2SO4, 100 ug/ml
BSA, 200 uM dNTPs, no more than 20-30 pmoles of each PCR primer, and no more
than 100 ng of template. 

We usually design our oligos with  10 G's and/or C's.  That is, we design 
our oligos such that they include no less than 10 G's or C's and/or
combination thereof.  Doing this makes them long enough for annealing tempera-
tures up around 60-68'C. When I PCRed 5kb, I simply used the 10X buffer
supplied with HotTub but the MgCl was brought to 2.5mM and 200uM dNTPs (my
oligos were both 40-mers). For our reaction, we also used 20-30 pmoles of each
primer and 100ng of template.  I haven't had a problem of excess template
inhibiting the enzyme but Wayne Barnes swears that it will.  However, I have
had excess primers screw up PCR reactions.  I let the elongation times go
upward of 7-10 mins at 40-50 cycles.  What else... 68'C for 1 min for anneal-
ing and 95'C 1 min melts - that's basically what I did to get a 5kb PCR
product.

[Second disclaimer - I don't work for Wayne Barnes! I'm in a separate Depart-
ment and division, but when I have PCR trouble, I'll seek his advice!]

Let me know if I can help any more... I hope that through this disorganized
mess there is something of use to the netters!

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