d/ddNTP ratios for Cycle sequencing with Taq
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Mon Dec 6 10:38:35 EST 1999
In article <82gi31$d5k$1 at bubba.NMSU.Edu>, KIM <dkim at hector.NMSU.Edu>
>This bias does not exist for T4 DNA polymerase (the basis
>of Sequenase) or Pol 1.
T7 DNA polymerase.
This bias DOES exist for E.coli pol I i.e. Klenow fragment as used in
sequencing many moons ago but does not exist in Sequenase, modified T7
DNA polymerase. T7 DNA pol is one of the few polA type sequences
published that incorporate ddNTPs efficiently. Mycobacterium pol Ialso
has this property. The Tabor & Richardson Tyr/Phe PNAS paper is a very
neat bit of work.
Roche had a poster at Thermophiles 98 that showed the isolation and
sequencing of polA sequences from unknown thermophiles with both the
ddNTP mutation and proof-reading activity. These were unknown in that
the genes were pulled by PCR from genomic DNA from the total population
of a hot spring. The bugs themselves were never isolated
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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