Automated dideoxy
DNA
sequencing with fluorescent ddNTPs
Synthesis of a new
strand from the DNA
template strand target proceeds with a reaction mixture including
the four
standard dNTPs and all four ddNTP, each labelled
with a
different fluorescent dye
(ddATP,
ddCTP,
ddGTP,
and ddATP).
Random
incorporation of the labelled ddNTPs
produces a series of DNA fragments
in which chain growth has been terminated at each successive
position,
each one nucleotide longer than the previous. Separation of the
fragments by size produces a sequencing ladder as a series of
colored
bands.
In an automated
DNA
sequencer, the fluorescent dye of each band is activated
by
a scanning laser as it
passes
a set
point at the bottom of the electrophoretic gel. The color of each
successive band is read by a fluorometer,
and
a
computer assembles these as a gel image,
which can be read from bottom to top, like a conventional
radioactively-labelled sequencing ladder. Multiple sequencing
reactions
on separate templates are run in parallel: the bands in each
ladder are
read as a separate electropherogram
or
chromatogram.