FR AB - Science Reference

ABRF 2001 ABSTRACTS
P1-S
Compatibility of the RapXtract Dye Terminator Removal Kit with
purification of sequencing reactions for the applied biosystems
Prism 3100 Capillary Sequencer.
A.L. Springer, L.R. Booth, K.A. Hughes, R.J. Kaiser, D.A. Spicer;
Prolinx, Inc., 22322 20th Avenue SE, Bothell, WA 98021
Successful automated DNA sequence analysis requires that sequencing reactions
be purified of excess unincorporated dye terminators. Common methods
for purifying sequencing reactions involve several steps and require
processes that are not easily automated. Prolinx, ® Inc. has developed the
unique RapXtract Dye Terminator Removal Kit for this application that is
rapid, easy to perform, yields high quality product and does not require modified
primers. The RapXtract kit uses a magnetic particle format to remove
dye terminator contaminants by mass action. The protocol has three steps:
1. Remove storage buffer from particles, 2. Add sequencing reactions and
mix, and 3. Remove purified extension products. The ABI PRISM 3100
Genetic Analyzer is a 16-capillary instrument that utilizes technology from
other ABI capillary sequencers in a medium-throughput format. RapXtractpurified
samples analyzed on the ABI PRISM 3100 yielded sequences with
good Phred quality scores and signal strength. The RapXtract kit has been
optimized to minimize the use of expensive sequencing reagents and can be
performed on reactions containing as little as 1 �l of Big Dye Reaction Mix
and as little as 5 �l total volume. The RapXtract kit has been used successfully
with double-stranded plasmid, PCR product or single-stranded DNA
templates. RapXtract purification can also be used for dye primer reactions.
The ease of use and versatility of the RapXtract kit make it a good choice for
manual or automated purification of sequencing reactions.
P3-T
Improved signal strength on ABI Prism 377 DNA Sequencer and
310 Genetic Analyzer by using a new sequencing run module.
S-M. Chen, H. Zielke, G. Amparo, S. Spurgeon; Applied Biosystems,
850 Lincoln Centre Dr., Foster City, CA 94404
In many sequencing labs DNA sample template quality varies greatly due to
the diverse sources of these samples and the different DNA preparation
methods used. This variable DNA template quality results in variable sequencing
data quality. Many low quality data are associated with low quality
or quantity of DNA templates. This frequently results in low signal strength
and low signal to noise ratio. A new sequencing module was developed for
the ABI PRISM 377 and 310 systems that will generate greater signal strength
during DATA collection. With the use of this module in data collection we
were able to improve signal to noise ratio and were able to improve success
rate in data analysis.
POSTER ABSTRACTS
188 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P2-M
Serial analysis of gene expression using the Applied Biosystems
capillary electrophoresis platforms.
K.M. Gunning, B. Nutter, A. Swei, J. Zon; Applied Biosystems,
850 Lincoln Centre Dr., Foster City, CA 94404
In today’s genomic environment, the draft completion of the human genome
has allowed researchers access to a wealth of information with respect to
gene identity and gene expression profiles. Scientists have developed novel
ways in which to elucidate this information and SAGE is deemed one of the
more comprehensive methods available for rapid, detailed analysis of large
numbers of cellular transcripts. To further enhance the rapidity at which one
can determine this global gene expression profile, one needs more efficient
and precise methods for analyzing these transcripts and for assessing their
abundance. Automated capillary electrophoresis provides a platform whereby
the sequence of these many transcripts can be determined with speed and
precision. The ABI PRISM. ® 3700 and the ABI PRISM. ® 3100, the most recent
addition to the CE family of platforms, provide the SAGE researcher with a
means by which they can determine quickly and reliably, the transcript
information necessary to build these gene expression profiles. These platforms
partnered with the uniformity of signal strength and length of read of
BigDye Terminators, are the labor saving answer to large scale transcript
analysis using the SAGE method. We will demonstrate the ease of use and
flexibility of these platforms as a preferred method for the sequence analysis
of ditags in the SAGE process.
P4-S
Two steps cycle-sequencing improves base ambiguities and signal
dropouts in certain BigDye DNA sequencing reactions.
L. Wen; San Diego State Univ., 5500 Campanile Drive, San Diego,
CA 92182-4614
The use of automated fluorescent DNA sequencer systems and PCR-based
DNA sequencing methods play an important role in the actual effort to
improve the efficiency of large-scale DNA analysis. While dideoxy-terminators
labeled with energy-transfer dyes (BigDye; PE/ABI) provide the most
versatile method of automated DNA sequencing, premature terminations
results in a substantially reduced reading length of the DNA sequence. Premature
terminations are usually evidenced by base ambiguities and are often
accompanied by diminished signal intensity after that point. I studied a twostep
protocol for Taq cycle sequencing using the ABI BigDye terminator for
reducing premature terminations in DNA sequences. I demonstrated that
combining the annealing step with the extension step at one temperature
(60�C) reduces premature terminations in DNA sequences that regularly contain
premature terminations when the three temperature steps is used. This
procedure can be significantly improved sequence quality in our core facility.
Sequence results from initial studies are still forthcoming and will be presented.