Biomedical Engineering Reference
In-Depth Information
the widespread availability of oligonucleotide syn-
thesizers. With this development, Sanger sequenc-
ing has been liberated from its attachment to the M13
cloning system; for example, polymerase chain
reaction (PCR)-amplified DNA segments can be
sequenced directly. One variant of the double-
stranded approach, often employed in automated
sequencing, is 'cycle sequencing'. This involves a
linear
amplification of the sequencing reaction,
using 25 cycles of denaturation, annealing of a
specific primer to one strand only and extension in
the presence of Taq DNA polymerase plus labelled
dideoxynucleotides. Alternatively, labelled primers
can be used with unlabelled dideoxynucleotides.
well suited to automation. To automate the process,
it is desirable to acquire sequence data in real time by
detecting the DNA bands within the gel during the
electrophoretic separation. However, this is not triv-
ial, as there are only about 10
−15
10
−16
moles of
DNA per band. The solution to the detection prob-
lem is to use fluorescence methods. In practice, the
fluorescent tags are attached to the chain-terminating
nucleotides. Each of the four dideoxynucleotides
carries a spectrally different fluorophore. The tag is
incorporated into the DNA molecule by the DNA
polymerase and accomplishes two operations in one
step: it terminates synthesis and it attaches the
fluorophore to the end of the molecule. Alternat-
ively, fluorescent primers can be used with non-
labelled dideoxynucleotides. By using four different
fluorescent dyes, it is possible to electrophorese
all four chain-terminating reactions together in
one lane of a sequencing gel. The DNA bands are
detected by their fluorescence as they electrophorese
past a detector (Fig. 7.6). If the detector is made to
−
Automated DNA sequencing
In manual sequencing, the DNA fragments are
radiolabelled in four chain-termination reactions,
separated on the sequencing gel in four lanes and
detected by autoradiography. This approach is not
V
−
Upper buffer
reservoir
Polyacrylamide
sequencing
gel
Electrophoresis
Fluorescently
labelled
DNA bands
V
+
Lower
buffer
reservoir
Scan line
Fluorescence detector
Sequence
AGCTTACGGACTAATGC
Fig. 7.6
Block diagram of an
automated DNA sequencer and
idealized representation of the
correspondence between fluorescence
in a single electrophoresis lane and
nucleotide sequence.
Dye 1
Dye 2
Dye 3
Dye 4
Time
Computer
