BIONET DNA Sequencing Survey

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Jun 17 08:33:00 EST 1993


In article <9306162222.AA08070 at net.bio.net>, AMCARTHU at UVVM.UVIC.CA (Andrew McArthur) writes...
> 
>         DNA Sequencing Survey: Taq vs. Sequenase
> 
>I while ago I put out a survey asking those researchers that
>have used BOTH Taq (or Vent) cycle sequencing and
>Sequenase sequencing to state their preferences.  Here are the
>results:
> 
>1. For those who definately prefer one over the other (9 of 14
>voters), six votes for Sequenase and three for Taq/Vent cycle
>sequencing.  However, those that came to prefer Sequenase
>were EXTREMELY emphatic.
> 
>2. For those who stated their preferences among the various
>cycle sequencing kits (5 of 14 voters), four voted for the
>Promega kit and 1 for the NEB kit.  Several other people
>mentioned their preference for Taq over Vent (purposes or
>methods not clearly stated).
> 
>3. For those who used different techniques for different
>purposes (5 of 14 voters), most used Sequenase for the majority
>of their projects but used Taq cycle sequencing in the case of
>lambda, cosmids, difficult-to-sequence templates, and templates
>in low concentration.  PCR products were sequenced by either
>method.  One voter stated M13 really only worked with Taq
>cycle sequencing while another made the claim it really only
>worked with Sequenase sequencing.

Sorry but you just hit a nerve on this one   :-)

All, and I mean ALL, of the major, large scale DNA sequencing labs
involved in mega-base sequencing as part of the Human Genome Project
are using fluoresce-labeled primers, Taq cycle sequencing, and
the ABI 373A's.  That's a fact and if we never run another radio-
labeled gel I'll be a happy camper.

As for radiolabeled sequencing, early on in the DNA sequencing world
everyone used Klenow.  With the publication of the Tabor and Richardson
paper on modified-T7 DNA polymerase and a deal struck with Promega,
"Sequenase" then came to replace Klenow as the enzyme of choice.
This is IMHO, mainly due to the work of Carl Fuller at Promega
who developed and tested the "kit".  It's pretty fool proof and
works for you're average user.  Almost everyone doing radiolabeled
sequencing seems to be using Sequenase.  There are some problems with
compressions (bands in all 4 lanes) but there are work-arounds.

Because of Patent constraints, there are several new thermo-stable
enzymes that have become available.  However, buy-in-large, the
HGP-large scale sequencing labs are still using PECetus's Amplitaq,
and are trying to negotiate a discount with the manufacturer.  Most
labs have played with the other enzymes but for various reasons, some
quite trivial, still use Taq polymerase for fluorescent-primer cycle
sequencing.

More recently, with the upgrades to the ABI 373A to a 5 filter wheel,
Sequenase is the enzyme of choice for the fluorescent-TERMINATORs
obtained from ABI.  That's because the phage enzyme "works better"
with ABI's new fluorescent-terminators than other DNA polymerases.

Lastly,  the statement:
	"One voter stated M13 really only worked with Taq
	 cycle sequencing while another made the claim it
	 really only worked with Sequenase sequencing."

is pure rubbish.  Protocols are available from multitudinous
(if there is such a word) sources, i.e. the primary literature,
suppliers poop sheets, etc., for successfully sequencing either
single stranded m13 or double stranded plasmids containing inserts
using any and all of the commercially available enzymes.

Cheers to one and all.............bruce
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 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
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