BIONET DNA Sequencing Survey

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sat Jun 19 08:26:00 EST 1993


In article <C8sJrp.yM at mentor.cc.purdue.edu>, pmiguel at bilbo.bio.purdue.edu writes...
>In article <17JUN199308330281 at aardvark.ucs.uoknor.edu>, broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
>>
>>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.
>>
>  Don't you mean USB, not Promega?

Yep and I've already corrected this error in a previous posting.

>  Also, aren't bands in all
>four lanes caused by enzyme "fall-off" (which does tend to be
>caused by sequence secondary structure, just like compression)?
> 

There is no evidence that it's "fall-off" or the enzyme just
mis-incorporates d/ddNTP's when it hits a stable sequence secondary
structure.  But you are correct about secondary structure being the 
culprit.

I have a tendency to think about bands in all 4 lanes as being due 
to "template compression" i.e.  secondary structure, since "product
compression" also is due to secondary structure ( fold back )
in the synthesized strand.

Cheers......bruce



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