Molecular Biology: DNA sequencing
Molecular Biology:
DNA sequencing
Author: Prof Marinda Oosthuizen
Licensed under a Creative Commons Attribution license.
AUTOMATED SEQUENCING
These days, the running and interpretation of sequencing gels is usually performed automatically. In
automated sequencing, fluorescent groups in the primer or in the ddNTPs replace the radioactive
label. The fluorescent groups are detected during the electrophoresis by laser irradiation and light
detectors.
Cycle Sequencing
One of the limitations of the standard chain termination method is that only a single labelled DNA
molecule is produced from each primer-template complex. The sensitivity of the method is therefore
limited by the amount of DNA template that can be used in the reaction. Cycle sequencing is a
method in which a small number of template DNA molecules are used over and over again to
generate the sequence ladder.
Most cycle sequencing chemistries these days use ddNTPs that are labelled with fluorescent groups
(dye-labelled terminators). The four ddNTPs are labelled with different fluorophores, each of which
has a different emission spectrum. The advantage of this is that the sequencing reaction can be
carried out in a single tube and analyzed in a single run. The cycle sequencing reaction mix includes
the template DNA, a primer, dNTPs, ddNTPs (each labelled with a different fluorophore) and a
thermostable DNA polymerase (usually a variant of Taq polymerase). The reaction mix is subject to
repeated rounds of denaturation, annealing and elongation, rather like a PCR. During the elongation
step, the thermostable DNA polymerase adds dNTPs or ddNTPs. The formation of the new DNA
strand is terminated upon the addition of a ddNTP. At the end of the cycles, multiple copies of every
possible fragment are present, each terminated by a ddNTP.
The sequencing reaction is purified and subject to electrophoresis in an automated sequencer. The
older automated sequencing machines (ABI 370, ABI 377, ALF, Hitachi, LiCor) had slab gels, but in
the newer machines the electrophoresis is carried out in a capillary containing a liquid polymer. After
each run the capillary is automatically emptied and refilled with the fluid polymer; the advantage of
this is that the capillaries can be reused many times. The ABI 310 has a single capillary, the 3100 and
3130XL have 16 capillaries while the 3700 model has 96!
Upon application of an electrical current, the fragments in the reaction mix migrate through the gel or
the polymer and are separated according to size. As the sequencing products reach the end of the gel
or the capillary, they are exposed to a laser beam which excites the fluorescent group and it emits
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Molecular Biology: DNA sequencing
light. The four different fluorophores emit light at different wavelengths which is detected by
photomultipliers. A computer program interprets the fluorescence that has been detected and
converts the pattern of fluorescent peaks obtained at the four different wavelengths into a nucleotide
sequence of 500 or even up to 800 nucleotides. The sequence is viewed in the form of an
electropherogram (Figure 4).
A very good animated illustration of automated sequence analysis, including both cycle sequencing
and sample electrophoresis, is available at http://www.dnalc.org/ddnalc/resources/animations.html .
Figure 4: Example of a portion of an electropherogram.
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