BIONET DNA Sequencing Survey

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Fri Jun 25 10:12:06 EST 1993


In article <17JUN199308330281 at aardvark.ucs.uoknor.edu>, broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
> In article <9306162222.AA08070 at net.bio.net>, AMCARTHU at UVVM.UVIC.CA (Andrew McArthur) writes...
>> 
>>         DNA Sequencing Survey: Taq vs. Sequenase

> 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.
>
I will admit that I can't quite follow this, so let me know if I am the only
one who lacks your "inside information".  When I buy Sequenase, it is from
United States Biochemical (USB), which is an excellent supplier with great
product support.  I also think Promega is pretty good, but I am not aware that
they had anything to do with Sequenase - they are a competitor of USB.
Some of the earliest sequencing was done with reverse transcriptase.  Also, we
used to do "quasi-end-labelling" with Klenow or RT and it gave wonderful
results.  I can't dig out my reference for it - it has been so long since I did
it, but this method was never adapted into a "kit" form because it had limited
utility.  You had to be sequencing with a primer which was followed closely by
a stretch of poly-anything.  We used the universal M13 primer which is followed
by four T's.  If you ran your first reaction with P32 dATP, but limiting
amounts of cold nucleotides, you could label up your new strands to pretty high
specific activity.  Then, you go on with the second step in which cold
nucleotides are added to extend.  I think this method was used by USB when they
developed the "two-step" system for Sequenase sequencing, but they never
pointed out that the 4 T's after the primer are contributing to the uniformity
of the bands in the same way they did with "quasi-end-labelling".  Enough
history!  I don't like automated sequencing and am waiting for people to
improve it before I waste any more time producing templates to be sequenced on
one of those instruments.  :)

Barbara Bugg
St. Jude Children's Research Hospital
Memphis, TN  USA> 
> Cheers to one and all.............bruce
>   - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
>  \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
>  /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
>   - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -  - - -



More information about the Methods mailing list