I have tried the enzyme that Epicentre Technologies calls
Sequitherm. Although we do not use it regularly in production, I have
done 600 or 700 reactions with it. The general conclusion is that it
seems to be much like taq, with two exceptions:
1) It seems to give somewhat more even peak heights- some bands
that are absent with taq are present with sequitherm
2) It seems to be somewhat more sensitive to contaminants in the
template DNA than taq- a larger percentage of the reactions that we do
with it fail to givwe good sequence
I would be _very_ interested in hearing what experiences other
people have had with other enzymes. I am particularly interested in
trying to find a thermostable pyrophosphatase, so that we could do to taq
what addition of PPase did for T7 Dna polymerase a la Tabor and
Richardson. I don't know if it would work, but suspect that it could make
a big difference... I have contacted 4 or 5 enzyme production companies,
but no one has gotten back to me yet with an PPase sample yet.
We are sequencing M13 ss templates purified in 96-well plates with
an unpublished protocol based on ethanol lysis of the phage coat-
basically Ethanol is used in place of phenol in the standard prep, and
everything is carried out in 96-well plates, with an affinity resin as
final cleanup. Samples are not as clean as phenol preps, but work well
with standard taq dye primer cycle sequencing.
Thanks for starting this discussion thread...
John Mulligan
Stanford Genomic Sequencing Center
415-725-7426
mulligan at genome.stanford.edu
(Forwarded by:)
Greg Lennon
Human Genome Center, L-452
Lawrence Livermore National Lab
Livermore, CA 94550 USA
Email: greg at mendel.llnl.gov
Phone: (510) 422-5711 Fax: (510) 423-3608
