cycle vs normal sequencing

Sasha Kraev bckraev at aeolus.ethz.ch
Thu Dec 22 12:07:57 EST 1994


Clemens,
I think ( it is my personal opinion, no connection to any vendor of kits,
instrumentation etc ) that the cycle sequencing eventually completely takes
over "regular" sequencing according to Sanger. The reasons are the following:

1. It works on a wider range of templates without changes in the protocol,
because it needs less template and is more forgiving of DNA quality

2. It runs at high temperature, thus avoiding many common problems, associated
with template secondary structure

3. It actually is more compatible with non-radiactive chemistries, than with
radioactive, e.g. with pre-labelled primers or terminators.

The current problem seems to be instrumentation, i.e. assembly of many small
PCR reactions, covered with oil is very cumbersome, compared to a typical
sequenase reaction run in a Terasaki plate. One may imagine special tiny tubes
(some are already on the market, but they need also a matching thermoblock,
e.g. you need also a new cycler or a slave block). When these new tubes are
invented, I believe nobody will spend time and effort getting 20-fold more
DNA needed for a regular reaction. Another problem of cycle sequencing is
that people often try to use existing primers out of the fridge to run
cycle sequencing. It is my impression that for good cycle sequencing primers
have to be a bit longer than usual, to allow two-step cycling ( 70/92 C),
The point here has something to do with the fact  that the efficiency of
a single cycle is much less than 2. So, when it will be found how one can
push( predictably ) efficiency as much as possible to this limit, 
cycle sequencing will be much more efficient than it is today and will become
THE sequencing method.

Cheers,
Sasha



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