Automated dideoxy DNA
sequencing with fluorescent ddNTPs
Replication of the DNA
template strand 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 DNAfragments
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
succesive 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.