help with seq. (why end label for cycle seq.)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Dec 14 10:01:07 EST 1994


Geoff Goellner writes:

> I've been using dideoxy thermal cycle sequencing, and have had trouble
> with "background" in 3 of the 4 lanes- pretty much all the way up and
> down the gel. I'm using P32-dCTP, Deep Vent (-), and a plasmid
> template. 

It sounds like you're starving the reactions for C because the chemical
concentration in the labelled nucleotide is too low to support extension
without stuttering every time you put in a C in any tube.  This is a
standard problem with sequencing methodology and every method has to
have a way to avoid it.  In old style Klenow sequencing, you had to
follow with a chase of all four triphosphates at high concentration to
extend the stalled products off the top of the ladder.  In the now
standard sequenase protocols, you put all the label in during a pre-
extension during which you don't care if the enzyme stalls because
you haven't added the terminators yet.

Cycle sequencing can't use either of these techniques, because there's
no way to get the products back on the template for a 2nd extension
step.  So almost nobody tries to incorporate during extension; instead
they end-label the primers.  If you absolutely have to label during 
extension, then you could lower the specific activity of the dCTP, thus
increasing its chemical concentration.  However, I don't recommend this
for general use because the conditions are likely to be very unforgiving.
There is a chase procedure with terminal transferase that could be 
tried (Fawcett and Bartlett, BioTechniques 9:46-48 (1990)).

Hope this helps.
Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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