FR AB - Science Reference

ABRF 2001 ABSTRACTS
P113-M
An automatable magnetic particle-based kit for the removal of
dye terminators.
D.A. Spicer, K.A. Hughes, R.J. Kaiser, A.L. Springer; Prolinx, Inc.,
22322 20th Avenue South East, Bothell, WA 98021
Prolinx ® Inc. has developed the RapXtract dye terminator removal system
that is fully automatable for high throughput purification of cycle sequencing
reactions. The kit is based on a novel magnetic particle format allowing
for rapid purification of up to 384 samples at one time, depending on the
robotic platform. The kit provides reproducible, high-quality results without
the need for modified primers. The RapXtract kit is easily automated, as it
does not require centrifugation, vacuum filtration, or multiple wash steps.
The kit has been automated on several platforms including the TECAN Genesis,
and the Tomtec Quadra 384 model 320.
P115-S
Systematic validation and optimization of capillary electrophoresis
for high performance genotyping and fragment analysis.
D. Shen, M. Minarik, A. Shuster, K. Pirkola, S. Gopalan, K. Dains,
M. Mahtani; Molecular Dynamics, 928 E. Arques Ave., Sunnyvale, CA 94085
Capillary electrophoresis systems have quickly replaced traditional fluorescent
slab gels for DNA analysis in many laboratory environments. The technology
transition has been abrupt and, on the up-side, has excitingly
advanced the Human Genome Project. On the down-side, the transition for
many labs has occurred without a clear understanding of the strengths and
weaknesses of the new technology, methods for troubleshooting, and experience
in setting benchmarks. We have spent the past year validating and
optimizing the capillary electrophoresis platform, the MegaBACE for high
throughput genotyping and fragment analysis. In the development of the system,
we have devised a set of metrics and guidelines that predict the system
performance.
We present data and benchmarks on variables including optical response,
dynamic range, sizing precision and reproducibility, resolution, throughput,
capillary and sample success rates, and other critical parameters of system
performance. These metrics offer a better understanding of proper system
function, and indices for perturbations of that function. We also provide data
on the performance of the software and the effects of modifications to the
workflow on the genotype outputs. In addition, we present a process control
system (including software and chemistry) that can be used to evaluate
the system performance or to test new methods and protocols before integrating
them in a production environment. In collaboration with others, we
will present data on the system’s performance for fragment analysis applications
including microsatellite genome scanning, AFLP analysis, SNP genotyping,
and differential display.
POSTER ABSTRACTS
216 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P114-T
Applications of an electric field-assisted capillary LC technique
and instrumentation for biological macromolecules analysis.
F.J. Yang, Y-H. Jou, C. Wu, C. Wu, Y.W. Hong; Micro-Tech Scientific,
140 South Wolfe Road, Sunnyvale, CA 94086
Recent rapid growth in the needs for biological macromolecules analysis and
drug discovery research has increased significantly the utilization of capillary
column separation techniques such as capillary column LC, capillary column
electrophoresis, and capillary column electro-chromatography etc. The
growth in the applications of capillary column separation techniques is also
driven by the commercialization of electro-spray and nano-spray LC-MS
interfacing techniques that allow mass spectra-analysis of biological macromolecules.
Major advantages of capillary column separation techniques are:
1). Improvement of detection limit by more than 2000 times in comparison
to that possible using conventional 4.6 mm id columns. The smaller the column
diameter, the lower the detection limits. It allows femto-mole detection
of trace amount of biological samples.
2). Direct interface to MS has greatly increased the applications of MS for biological
sampler analysis.
3). It reduces solvent consumption by more than 2000 times.
4). 2D LC-MS can be easily configured for rapid sample clean up, de-salt,
matrix elimination, and sample concentrating. It allows fast capillary LC-MS
for high through put sample analysis.
5). Capillary columns are common to capillary LC, CE, and CEC, it facilitates
an unified methodology and instrumentation for harness the advantages of
capillary column separation utilizing both differential electrophoretic migration
of charge molecules and the differential mobility of the sample molecular
zones carried by either electro-osmosis flow or hydrodynamic flow.
This presentation will discuss the concept of an unified capillary column separation
methodology and the design of the instrumentation that can perform conventional
CE and CEC. It can also be utilized for both low pressure and high
pressure gradient (pH, buffer strength, and organic composition gradient) CE
and CEC applications. Examples for the applications of the unified capillary separation
system in the electrical- field assisted high pressure capillary gradient LC
applications for biological macromolecules analysis will also be presented.
P116-M
Long sequence readlengths with the MegaBACE 1000
DNA Analysis System.
J. Ellerbrock1, M. Reagin1, A. Mamone2, J. Nelson1, B. McArdle1, C. Fuller1; 1AP Biotech, 800 Centennial Ave., Piscataway, NJ 08855,
2Molecular Dynamics
MegaBACE 1000 DNA Analysis System is a high-throughput, fluorescencebased
DNA sequencer that utilizes capillary electrophoresis with 96 capillaries
operating in parallel. The system automates gel matrix replacement, sample
injection, DNA separation, and data analysis with integrating software.
The sensitivity of DYEnamic ET energy transfer terminators and the superior
resolving power of linear polyacrylamide (LPA) separation matrix allow
detection over a broader range of template amounts. Likewise, the robust
performance of Thermo Sequenase II DNA polymerase improves success
rates.
The MegaBACE 1000 has the ability to sequence 96 samples in just 2 hours,
perform up to 9 runs per day, and can be used in both high-throughput and
core DNA analysis facilities. In high-throughput facilities it can produce as
much as 500,000 raw sequence bases a day. We have developed protocols
that allow reads of over 1000 bases ideally suited for final overnight runs (4–6
hours) when such sustained high-throughput is not required. We will present
a variety of examples and protocols to obtain optimal results on the system.
Data will be presented showing long DNA readlengths on a variety of different
templates (BAC, plasmid, M13, high GC, or PCR product) using the
DYEnamic ET terminator cycle sequencing kit and the MegaBACE 1000 system.