Tom,
We were recently plunged into a project involving heterozygote detection
and began using our ABI 377=92s and the Prism dye terminator kits. The
results were a little mixed. We could detect the polymorphic positions
but only if we were looking for them. Since we were looking at known
polymorphisms this wasn=92t a major problem but the software was
unreliable in detecting them. We also tried some custom dye primers
(about $2000 a pop) but were still not satisfied with the results. What
we eventually opted for as a more standard approach was using
conventional isotopic sequencing.
Amersham has recently release a 33P terminator kit using
Thermosequenase. By using the labeled terminators the ghost bands that
are often see with isotopic sequencing and which would complicate
heterozygote detection are avoided (these sequencing ladders are really
clean and just beautiful to look at -- the heterozygotes just pop right
out at you). Also with the low energy and short half-life of 33P a lot
of the concerns over using radioisotopes are not really an issue. As
for hardware we chose the sequencers manufactured by Genomix. These are
high performance electrophoresis units and a real joy to work with
compared to other manual sequencing apparatus I=92ve used in the past.=20
The gels are dried in the unit right onto the back plate so there=92s no
messing around with huge gels floating around in a fixing bath. The
units can also be run at up to 70 C so there are virtually no
compression problems. In fact the company boasts that there is nothing
that they can=92t sequence through. They also have a "scrubber" unit
which removes the isotope from the buffer.
With respect to labor considerations we=92ve found that this setup is
comparable to automated sequencing. The most time consuming part is in
setting up the reactions (the standard 4 reactions for each template)
but this would be the same if dye primers were used in an automated
system. The only draw back is with throughput. The ABI=92s can
definitely put through more samples in a single run but then again you
can do two runs on the Genomix sequencers in the time it takes to do a
single run on the ABI=92s so it sort of balances out. If you are just
setting things up something else to consider is the cost involved. The
Genomix sequencers are only a fraction of the cost of an ABI. For the
price of a single 377 you could buy a number of Genomix units along with
a scanner and software for analyzing the autorads. With respect to
reagent cost it costs about $8 per template on the ABI=92s and about half
that with the Amersham kits.
After starting this project we looked into a number of technologies and
in our case I think we made the best decision. We=92ve been doing
automated sequencing for a number of years now and I never thought I=92d
be supporting the manual approach again but there you have it. We still
do the bulk of our sequencing on the ABI=92s but if you are primarily
interested in heterozygote detection I would give this approach some
serious consideration.
I hope this helps somewhat with your decision.
Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada
Ph#: (613) 941-6441
FAX#: (613) 957-1358
E-mail: styler at hpb.hwc.ca
