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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.
P117-T New separation medium for the ABI Prism ® 3700 DNA Analyzer and ABI Prism ® 3100 Genetic Analyzer. B.F. Johnson1, K.O. Voss2, J.N. Tian2, J.A. Fisher2; 1Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, 2Applied Biosystems, Foster City, CA A new separation medium for the ABI PRISM ® 3700 DNA Analyzer and ABI PRISM ® 3100 Genetic Analyzer has been developed. The new polymer formulation has a viscosity similar POP-5 and POP-6 formulations previously released for DNA separations on our capillary instruments and consequently has similar capillary filling times and sheath flow characteristics. High quality separations can still be obtained with bare capillaries with a minimum of 100 separations before cleaning or replacement of the capillary array. Compared to POP-5 separation medium using the default run conditions for POP-5 on the ABI PRISM ® 3700 DNA Analyzer, the new separation medium results in a 20% decrease in electrophoretic separation time while increasing one-base peak resolution by 100 bases. On the ABI PRISM ® 3700 DNA Analyzer with a 50 cm array the new polymer formulation under these conditions results in an average length of read of 850 bases at 10 runs per day. On the ABI PRISM ® 3100 Genetic Analyzer the new polymer formulation results in an average length of read of 825 bases in a 50 cm array at 16 runs per day. Basecalling length of reads obtained from ABI PRISM ® Sequencing Analysis (1% error) and from TraceTuner 1.1 analysis software will be compared at different separation voltages, distances, and temperatures. Run parameters that yield the maximum length of read or maximum number of runs per day will be presented. P119-M Converting from slab gel to capillary electrophoresis: a user’s guide for genotyping. C.L. Brown, B.F. Johnson, C. Wike, K. Roy, A. Wheaton, Y. Wang, N. Caffo; Applied Biosystems, 850 Lincoln Centre Drive, MS 404-1, Foster City, CA 94404 Capillary electrophoresis has rapidly become the platform of choice for human disease research, clinical diagnostics and population genetics studies due to its ease of operation, automation and increased throughput. Historically, most protocols were developed on slab gel instruments due to their flexibility, high throughput and consistency, yet required considerable cost and labor. The ABI PRISM ® 3100 Genetic Analyzer, a 16-capillary electrophoresis instrument, was introduced to match the throughput and reproducibility of slab gels while offering lower run costs, less human intervention and greater sensitivity. Laboratories considering the conversion from slab gel systems should be mindful of a few distinctions between these two platforms. Herein, we will discuss modified sample preparation, fragment mobility differences and suggestions for multiplexing schemes. We will also highlight enhancements to data analysis, which reduce existing rate limiting steps. POSTER ABSTRACTS ABRF 2001 ABSTRACTS P118-S Modifications to 96-well DNA preparation kits for the MegaBACE 1000. R. Dhulipala1, B. McArdle1, A. Mamone2; 1AP Biotech, 800 Centennial Ave., Piscataway, NJ 08855, 2Molecular Dynamics With the introduction of high-throughput DNA sequencing instruments, methods for simultaneous preparation of template samples have come into common usage. Several groups have developed plasmid preparation methods enabling the user to easily decrease the time to prepare 96 DNA samples and thus increase sequencing throughput. The majority of these kits are based upon traditional alkaline lysis of bacterial culture yet differ in their method of purification of DNA. We have evaluated several of these methods for preparing templates for the MegaBACE 1000 capillary instrument. We find that results obtained with capillary sequencing instruments are dependent upon the quality and amount of the starting template. Hence, results might vary greatly due to the method of DNA preparation. We have found that most of the preparation methods and kits are configured to optimize DNA yield, not well-to-well consistency. Fortunately, the DNA yields are greater than necessary and simple modifications to the standard protocols can be made that improve consistency without reducing yields significantly. Each purification method is evaluated for amount and consistency of DNA yield, and sequencing performance using DYEnamic ET terminators and the MegaBACE 1000. In addition, we make recommendations for each method to achieve optimal success rates with the system. P120-T Sequencing reaction purification of BAC DNA using a combination of molecular weight cut-off membrane and a new grade of Sephadex G-50. R. Dhulipala, A. Kumar; AP Biotech, 800 Centennial Ave., Piscataway, NJ 08855 The purification of DNA sequencing reactions by ethanol precipitation or gel filtration prior to the loading of automated fluorescent DNA sequencing instruments is a standard technique for the removal of unincorporated dye terminators. One standard gel filtration method that we have developed into a ready-to-use 96-well format with pre-swollen DNA Grade Sephadex G-50 is AutoSeq96 (please see our companion poster). AutoSeq96 purification is six times faster than ethanol precipitation, is easy to use, and minimizes handling because samples are purified directly into 96-well collection plates. However, one aspect of DNA sequencing reaction purification that has remained cumbersome for some types of samples containing large DNA constructs is the additional removal of template DNA. We have developed a new type of gel filtration method for purifying DNA sequencing reactions using the basic format of the AutoSeq96 plate but with the added benefit of template removal. Elimination of template DNA prior to electrophoresis can both increase the overall success rate of the reactions and improve read lengths for large plasmids and BACs. This new gel filtration method, called PureSeq96, has been optimized in 96-well format using a high molecular weight cut-off membrane (MWCOM) combined with preswollen low conductivity Capillary Electrophoresis Grade Sephadex G-50. The combination of the unique properties of the MWCOM and this new grade of G-50 are expected to improve sequencing success rates of both large construct plasmids and BACs using MegaBACE 1000. Described in this poster are the results of our studies and some of the development issues considered during the design of this gel filtration method for DNA sequencing reaction purification. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 217
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