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Automated fluorescent DNA analysis
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specific manner (2). In a typical sequencing experiment,
four separate reactions are performed, each containing
primer, DNA template, polymerase, and the four deoxynucleotides (dNTPs). By adding a different chainterminating ddNTP to each reaction, a nested set of
oligonucleotide products is generated, each terminating
with the specific ddNTP present in that reaction and thus
representing the occurrence of the corresponding dNTP
in the sequence. When the products of the four reactions
are electrophoresed side-by-side, the DNA products in
each lane migrate according to their size. By using
labelled primers, dNTPs, or ddNTPs, the positions of
the separated products can be detected and the sequence
deduced. When the label is a fluorescent dye, automated
analysis of the data becomes possible.
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Dye-terminator sequencing
In dye-terminator sequencing, a fluorescent label is
incorporated into DNA sequencing reaction products by
using dye-labelled ddNTP terminators. Both single- and
four-colour fluorescent dye chemistries are available,
depending upon the type of sequencing instrument used.
When each ddNTP is tagged with a different fluorescent
dye, a single reaction mixture can be used, and reaction
set-up is greatly simplified. Since prematurely terminated
chains are not labelled in dye-terminator sequencing, stop
artefacts and most background bands are eliminated.
Automated sequencing initially offered a non-radioactive
method for automated data collection. Improvements in
enzymology, dye chemistry, and instrumentation have
advanced the technology to the point where automated
fluorescent DNA analysis provides the high-throughput
capacity demanded by modern DNA sequencing and
analysis laboratories.
Energy transfer dyes
The recent introduction of energy transfer (ET) dyes
(3, 4) for improved detection in automated DNA
sequencing has resulted in development of a line of
exceptionally robust sequencing products with improved
signal strengths and uniform peaks. In 1995, Amersham
Biosciences introduced DYEnamic™ ET primers
for use with ABI™ sequencing instruments. DYEnamic
ET Terminator Cycle Sequencing Kit was launched in
1998. Depending upon the specific product, either
primer or terminator components of the sequencing
reaction are labelled with two fluorescent dyes.
The 5386-bp genome of bacteriophage φX-174 was first
sequenced more than 20 years ago by Sanger and his
colleagues who used 32P-labelled nucleotides to accomplish the task (1). During the following two decades, as
the scope of sequencing projects sharply expanded, it
became apparent that radioactive methods, although
highly sensitive, were limited in their potential for high
throughput. Alternative methods, amenable to automation, were thus sought to keep pace with laboratory
demands, and automation based on fluorescence
emerged as the method of choice. For this shift from
radioactive to fluorescent methods to occur, without loss
in sensitivity, improvements in sequencing instrumentation, enzymology, and dye chemistry were necessary.
In DYEnamic ET Terminator Cycle Sequencing Kit,
each of the four chain terminators is tagged with a
fluorescein donor dye and a rhodamine acceptor dye
(rhodamine-6-G, rhodamine 110, tetramethyl rhodamine, or rhodamine-X). Laser light is absorbed by
the donor and transferred to the acceptor dye which
then emits light at its characteristic wavelength for
detection and identification of the specific terminator.
Dideoxy sequencing—general background
Dideoxy sequencing is based on the ability of dideoxynucleotides (ddNTPs) to terminate primer extension
reactions catalysed by a DNA polymerase in a sequence-
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previously available sequencing enzymes. Thermo
Sequenase II DNA polymerase is currently the best
enzyme available for dye terminator sequencing (Fig 2).
Fig 1. 200 ng of M13mp18 DNA was sequenced with DYEnamic ET
Terminator Kit on ABI 377 sequencer according to the recommended
protocol with 1 min extension time at 60 °C.
Energy transfer leads to a more efficient excitation of
the acceptor dyes than direct excitation by the laser,
resulting in exceptional sensitivity and signal strengths
several fold greater than those obtained with conventional single-dye labelled molecules (Fig 1) (3).
Better polymerases—better results
Fig 2. 400 ng of pUC19 DNA was sequenced with Thermo Sequenase II
Dye Terminator Sequencing Kit on ABI 373 sequencer according to
the recommended protocol.
Although it has the advantage of being automated,
fluorescent detection is generally less sensitive than
radioactive detection. To compensate and improve
signal strength, it is necessary to either increase the
amount of DNA template used in the reaction or to
increase the amount of sequencing reaction product
through cycle sequencing.
Platforms for automated DNA sequencing
and analysis
In the late 1980s, Amersham Biosciences
launched ALF™ DNA Analysis System, an automated
DNA sequencer. In response to demands for improved
instrumentation with higher capacity, new automated
sequencing platforms have emerged: ALFexpress™ II and
SEQ4X4™ for medium- to high-throughput sequencing
with slab gels and MegaBACE™ 1000* for productionlevel sequencing using capillaries.
T7 Sequenase™ DNA Polymerase (5, 6), long recognized
as the “gold standard” in dideoxy sequencing enzymes,
readily incorporates both dNTPs and ddNTPs and yields
highly uniform signal intensities on sequencing gels.
Unfortunately, this polymerase is not stable at 95 °C
and cannot be used for cycle sequencing. In 1995, Tabor
and Richardson (7) identified the region in T7 DNA
polymerase that, when substituted into E. coli DNA
Pol I, makes the E. coli enzyme as non-discriminatory
towards ddNTPs as T7 DNA polymerase. Using this
information, Amersham Biosciences developed
Thermo Sequenase™ DNA Polymerase (8, 9), a thermostable enzyme that readily accepts ddNTPs and has no
5'-3' nuclease activity. Ideal for cycle sequencing, the
enzyme generates bands of uniform intensity for
improved base-calling by automated sequencers.
Automated sequencing with ALFexpress II DNA
Analysis System
ALFexpress II DNA Analysis System (Fig 3) is a fully
automated, user-friendly system supported by a wide
range of software packages, novel gel products, sequencing reagents, convenient application kits, and remote
diagnostics. With its many support products, ALFexpress II
is applicable for almost every type of sequencing and
fragment analysis, including mutation detection,
microsatellite and linkage analysis, and HLA typing.
For sequencing, ALFexpress II uses dideoxy chemistry
in a one-dye, four-lane format. When electrophoresed,
DNA products labelled with the carbocyanine dye
Cy™ 5 are detected as they pass through a laser beam at
Further modifications resulted in Thermo Sequenase II,
which readily incorporates dITP for improved performance on GC-rich templates and shorter cycle times.
Thermo Sequenase II exhibits higher processivity, higher
salt tolerance and more strand-displacement activity than
*Not available in all countries. Please contact your local Amersham
Biosciences representative for additional information.
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from ALFexpress II and performs qualitative and quantitative analysis of DNA fragments such as micro- and
minisatellites. Mutation Analyser software analyses
sequence data to detect specific sequence aberrations
in genomic DNA or cDNA/mRNA.
Automated DNA sequencing with SEQ4X4 Personal
Sequencing System
The compact SEQ4X4 instrument (Fig 4) is designed
for rapid electrophoretic separation and detection of
four sets of sequencing reaction products using Thermo
Sequenase and either Cy5.5-labelled terminators or
primers. From gel casting to data export, SEQ4X4
requires only 60 min to generate four sequences, each
300 or more bases in length.
Fig 3. ALFexpress II DNA Analysis System is applicable for almost
every type of sequencing and fragment analysis, including mutation
detection, microsatellite and linkage analysis, and HLA typing.
Gels can be cast in seconds using pre-filled RapidGel™
cartridges and QuickFill gel plates and polymerized in
minutes with UV light in the RapidSet™ gel polymerizer.
QuickFill plates require minimal preparation, no
disassembly, and can be discarded after a single use.
a fixed point in the gel. Light from the fixed laser excites
the Cy5 label simultaneously in up to 40 lanes of the gel
(corresponding to 10 sequencing templates or 40 fragment
analysis samples). The resulting fluorescence is detected
by a linear array of photodiodes that generates a signal
that is sent to the system’s computer. The signals from
each set of four lanes are then superimposed to create
the full sequence for each sample.
With no moving parts in the optical system, the sequencer
has a high signal-to-noise ratio and requires minimal
servicing. SEQ4X4 is controlled by software operating
under Windows NT™ 4.0. Run parameters can be set at
the start of a run with raw data collected and displayed
in real time.
ALFexpress II is controlled by ALFwin™ Control
Module within ALFwin Sequence Analyser or ALFwin
Fragment Analyser software. Each software package
allows the user to control the run using specific casebooks that contain all experimental run conditions,
sample information, and post-run actions. Results can
be monitored in real time on the screen, subjected to
analysis, and presented in a variety of formats either
on-screen or in customized printed reports. Several
export formats are also available for further analysis
of the data in other software applications.
The availability of pre-mixed photo-polymerizable gel
solutions means that uniform and consistent gels can be
cast and ready for use in minutes rather than hours.
ReproGel™ gel solutions, especially developed for
ALFexpress II, contain a novel polymerization initiator
that works when exposed to UV light in ReproSet™ UV
box. After 10 min of illumination, the gel is ready to use.
Fig 4. SEQ4X4 Personal Sequencing System delivers sequencing results
up to five times faster than radioactive sequencing from start to finish.
Shown above (from left to right) are SEQ4X4 Personal Sequencer, Fill
Fixture/Fill Gun, and RapidSet Gel Polymerizer.
A range of software packages, all using data generated
by ALFexpress II, supports the versatility of the system
for various DNA sequence and fragment analyses.
HLA SequiTyper™ compares sequence data generated
by ALFexpress II with a set of master sequences to
automatically determine the HLA type of class I and II
genes. For automated allele identification and genotyping, AlleleLinks™ software reads raw data directly
Base-calling is either automatic or semi-automatic if
the user defines start and stop points and peak interval.
With either method, raw data can be aligned and
converted to sequence in a matter of minutes, and the
sequence can be edited without affecting the raw data.
Additional options in the analysis menu simplify
detection of heterozygotes. Sequences can be saved as
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standard chromatograph format (SCF) files or as text
files for analysis by other software packages or for
database searches. Several printing options allow
customized reports and presentations.
Automated DNA sequencing with MegaBACE 1000
Designed for production-scale DNA analysis,
MegaBACE 1000 (Fig 5) is state-of-the-art technology
for genomic sequencing. The instrument integrates the
expertise of Molecular Dynamics and Amersham
Biosciences in sequencing instrumentation, software, and reagents, and is now used in major commercial
and academic sequencing centres worldwide.
Fig 6. Sequence data generated by MegaBACE 1000 Sequencing System.
Sequencing reactions were performed using DYEnamic ET Primers
and Thermo Sequenase DNA Polymerase.
In contrast to traditional slab gel systems, MegaBACE
utilizes 96 capillary columns, operating in parallel, to
separate, detect, and analyse fluorescently labelled DNA
fragments. Capillaries, pre-assembled in arrays of 16,
are designed for easy replacement and durability. The
capillaries are expected to last at least 100 sequencing
runs. Gel matrix replacement, sample injection, DNA
separation, and data analysis are all automated.
A complete range of fully optimized reagents is available
for use with MegaBACE 1000, including four-colour
dye-labelled terminator and primer products. Read
lengths of 500 bp or greater are routinely achieved
with MegaBACE 1000 LPA Matrix. With its capacity
for long read lengths, MegaBACE 1000 is equally
suited for both production and finishing, making it the
most highly automated, yet versatile system available.
MegaBACE 1000 accepts samples in a 96-well format
and automatically processes 96 samples per sequencing
run with a total turnaround time of less than 2 h. Since
each run requires less than 10 min of hands-on time,
more than 1 100 sequencing templates can be analysed
in a single 24 h period. The user interface permits
Summary
To meet the demands for high-throughput DNA sequencing and analysis in the 1990s, radioactive-based
methodology has largely given way to fluorescence-based
procedures that are highly sensitive and easily automated.
Through advances in enzymology, dye chemistry, and
instrumentation, automated sequencing has moved
beyond the realm of automated data collection and
into complete system automation from gel pouring to
data analysis.
References
1. Sanger, F. et al., Nature 265, 687–695 (1977).
2. Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74,
5463–5467 (1977).
3. Ju, J. et al., Proc. Natl. Acad. Sci. USA 92,
4347–4351 (1995).
4. Ju, J. et al., Anal. Biochem. 231, 131–140 (1995).
5. Tabor, S. and Richardson, C. C., Proc. Natl. Acad.
Sci. USA 84, 4767–4771 (1987).
6. Tabor, S. and Richardson, C. C., J. Biol. Chem. 264,
6447–6458 (1989).
7. Tabor, S. and Richardson, C. C., Proc. Natl. Acad.
Sci. USA 92, 6339–6343 (1995).
8. Reeve, M. A. and Fuller, C. W., Nature 376,
796–797 (1995).
9. Vander Horn, P. B. et al., BioTechniques 22,
758–765 (1997).
Fig 5. MegaBACE 1000 Sequencing System automates gel matrix
replacement, sample injection, electrophoresis and data analysis.
Sequences of over 800 bases can be obtained in less than 2 h.
simultaneous real-time viewing of all 96 capillaries,
and sequence data from individual lanes may be selected
and represented graphically in separate windows during
the run (Fig 6).
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