Q: Sequencing GC rich regions // Looking for sequencing kit/method using a LABELLED TERMINATOR (ddNTP)

David Micklem drm21 at mole.bio.cam.ac.uk
Tue Sep 3 08:04:24 EST 1996


In article <50fjm3$db1 at sparcserver.lrz-muenchen.de>,
diabetes at lrz.uni-muenchen.de wrote:

>Hi folks! Sorry for the long subject.
<snip>
>Now I want to try another way of radiolabelling the sequencing
>products by using radioactive ddNTPs in order to see only the
>correctly terminated DNA transcripts on the gel. But not using 33P.
>32P or 35S is OK. (Maybe we're kind of stoneage here?)
>Who can help or give hints? YOUR opinion is very appreciated!
>
>please mail and post your reply.
>
>Wolfgang Schechinger

Hi Wolfgang,

If I understand you correctly, then you think that the bands you are
getting are the result of non-ddNTP termination.  If this is the case, you
could try one of three methods that I have heard alluded to on this
newsgroup recently (I haven't got round to trying them yet).  They all
sound simpler (and cooler!) than termination-labelling your reactions:

1) Daniel Mytelka and Michael Chamberlain NAR1996, vol24, pp2774-2781 -
they find that addition of 2M betaine to the termination reactions can
cure T7 polymerase pauses.

2) Addition of a terminal transferase reaction step after
extension/termination: the terminal transferase will extend only chains
NOT terminated with a dideoxy nucleotide.  Incorrectly terminated chains
get so large that they no longer interfere (or maybe its just that their
size distribution is large enough that they don't form a visible band...).
I'm sorry, I don't have the ref, or a detailed protocol.

3) Felix Guerrero posted a Klenow co-sequencing ref (Biotechniques Vol 17,
pg. 286 (1994)), which I think works much the same way as terminal
transferase, but is simpler.  He kindly posted the protocol:

>The Klenow co-sequencing is surprisingly very simple. I use the standard
>Sequenase Ver. 2.0 protocol with the only change being that after adding
>the Sequenase enzyme to the DNA-annealled primer/buffer solution, one adds
>1 unit of Klenow. Then the only difference is to allow the termination
>reaction step to proceed at 37 degrees C for 45 minutes instead of the
>usual 2-4 minutes.

>Pretty easy and I think there is a noticeable improvement in band
>uniformity from difficult templates. At times, I have had to resort to
>dITP sequencing, but now I add the Klenow routinely!

I'm going to try this method first, next time I have a problem template. 
I can't see any reason why the Klenow shouldn't be incorporated into the
master mix that I distribute, rather than added seperately.

I'd be very interested to hear whether/how you  manage to solve your problem.

Cheers,

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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