In the past decade, advancements in DNA analysis
technologies have had an enormous impact on the
progress of life science research and have fueled
the genomics revolution. From the genome assembly
of the Severe Acute Respiratory Syndrome
(SARS) virus to HapMap construction, DNA
analysis continues to be a vital technical approach
for advancing life science research. However, the
cost of genetic analysis has historically limited
both the number of sites with DNA analyzers and
the scope of DNA analysis projects. The latest generation
of powerful capillary electrophoresis (CE)
DNA analyzers are capable of reducing genetic
analysis project costs by 50% or more compared
with previous technologies, while offering greater
data quality, enhanced laboratory productivity, and
greater flexibility.
The latest generation of CE instruments, specifically
the 48-capillary 3730 DNA analyzer and 96-capillary 3730xl DNA analyzer (Applied
Biosystems, Foster City, CA), offer a much
higher level of performance than previous-generation
DNA analyzers, and expand the range of
possible applications for the technology. The
3730 series platform combines industry-standard
reagents, advanced sample tracking, and automation
with innovative optics and capillary design.
The enhanced optical design provides a higher
signal-to-noise ratio and a more uniform signal
profile across the array. This design, combined
with a POP-7™ polymer, enables longer read
lengths and improved color balance for handling
fragment analysis samples. Automation features
include an integrated plate stacker, internal bar-code
reader, and on-board polymer delivery pump
(PDP) for highly reliable, unattended operation
for up to 48 hr.
Novel assay designs, coupled with instrument
advancements and new software, have expanded
the versatility of DNA analysis instruments for the
next generation of genomic research. In addition
to de novo sequencing and fragment analysis, examples
of new applications for use on the systems
include medical sequencing, or resequencing, and
genotyping to identify genetic polymorphisms of
predictive significance.
CE has remained the gold standard technology for
DNA analysis by providing high data quality, application
and read-length flexibility, and project cost
savings. As a result, the latest generation of CE
instruments from Applied Biosystems are routinely
used by institutions worldwide, including academic research laboratories, core and service laboratories,
large genome centers, and biotechnology and pharmaceutical
companies.
The Genome Sequencing Center at the Washington
University School of Medicine (St. Louis, MO) was
a major contributor to the Human Genome Project
and is an internationally esteemed research institution.
According to Dr. Elaine Mardis, Associate
Professor in Genetics and Director of Technology
Development, the Genome Sequencing Center used
ABI PRISM® 3700 DNA analyzers (Applied
Biosystems) for the bulk of the sequencing work on
the human and mouse genomes, but has since moved
most of its work to the 3730xl DNA analyzers.
According to Dr. Mardis, because the 3730xl systems
are much more productive than previous-generation
3700 CE instruments, the Center needs
fewer of them, and also requires fewer technicians
and reagents. The Center has begun using the
instrument exclusively for its genome sequencing
projects because of the substantial cost savings that
it provides.
Over the years, the Genome Sequencing Center has
continually invested in advanced technology in order
to reduce costs while meeting increasing demand. The
Center recently used the analyzer to sequence the
chimpanzee and chicken genomes, and work is currently
under way on two species of Drosophila, the planarian
worm, and three additional species of
Caenorhabditis (roundworm).
One of the strongest advantages of the instrument is
reduced reagent usage from the previous-generation
instruments, which substantially reduces sequencing
costs. Further, increased automation has contributed
to significant increases in reliability and productivity
for users of the analyzer. The instrument can be
set up to run for two or three days without human
intervention. The Center has purchased more
3730xl analyzers, and the number of technicians
required for sequencing has been substantially
reduced, even as its throughput has significantly
risen. Taking into account the reduced reagents,
personnel, and maintenance required by the
machine and its longer read lengths, costs have
dropped by greater than 50% per read.
Washington University Core
Laboratory
The Washington University Protein and Nucleic
Acid Chemistry Laboratory (PNACL) uses the 48-capillary 3730 DNA analyzer for the majority of
sequencing projects. This organization functions as
an academic core laboratory, providing services on a
fee basis to researchers at the Washington University
School of Medicine and undergraduate campus as
well as from other universities and biotechnology
companies. Like most core laboratories, the PNACL
processes a greater variety of samples and sample
concentrations prepared under a myriad of different
methods, and appreciates the benefits of a high-throughput
instrument capable of accurately and
easily tracking and processing every sample type with
high sensitivity, minimal failures, and little or no
drop-off in data quality.
Mr. Timothy Lehnhoff, Laboratory Manager for
PNACL, explained that one of the key features that
attracted the laboratory to the 3730 DNA analyzer is
that one plate of sequencing and another plate of
genotyping can be set up to run one right after
another, automatically. The laboratory is achieving
99% accuracy on fragments of 800–900 base pairs
with the analyzer, a level that was previously reached
only on fragments below 600 bp on the Applied
Biosystems 3100 genetic analyzer. Reducing the
number of sequence reactions that need to be rerun
significantly lowers the cost per sample. For example,
the laboratory can now usually sequence a 1000-base
insert forward and reverse with full overlap in two, at
the most three, sequence reactions. Most of the laboratory’s
customers are operating on NIH grants at
reduced levels of funding, and therefore a reduction
in sequencing costs is most welcome.
The laboratory’s growth has occurred mainly in
genotyping in recent years as researchers begin to
apply the human genome to the practical work of
identifying alleles and mutations. The much faster
throughput of the instrument has made it possible to offer price reductions, particularly to users who bring
the laboratory higher volumes of work, such as 96
samples in a 96-well plate. The 3730 analyzer also
has made it possible to reduce normal turnaround
time for up to 50 samples from 48 hr to 24, which
helps improve the laboratory’s competitive position.
The instrument also provides substantial cost savings
over the instruments previously used at the laboratory,
because its integrated autosampler automatically
loads all 48 samples simultaneously, substantially
reducing the time required for loading and
injection of samples. The higher data quality and
sensitivity of the instrument provide additional cost
savings by supplying longer read lengths across a
variety of sample concentrations.
The fact that the Washington University Core
Laboratory uses the 3730 DNA analyzer while the
Genome Sequencing Center uses a 3730xl analyzer
highlights the potential different advantages of the
two units. The 96-capillary 3730xl analyzer is
designed for high-throughput sequencing, large-volume
projects, or for those who need the flexibility
to offer services for a high volume of samples.
The 48-capillary 3730 analyzer is intended for
users in a high-throughput setting who have
slightly lower throughput needs than provided by
the 96-capillary system, and who appreciate the
value of a somewhat lower-priced system, but still
wish to invest in the benefits of the latest high-throughput
technology.