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ABI PRISM 3100 Genetic Analyzer:A Fully Automated and High-Performance16-Capillary Electrophoresis System forFragment Analysis ApplicationsYiwen Wang, Liwei Qi, Kitland Louie, Dong Wei,Xiaohui Chen, Ruben Pingue, Cydne Holt, BenJohnson, Eric Nordman, Jing Tian, Ariana Wheaton,Nathan Caffo and Penny DongApplied Biosystems850 Lincoln Centre DriveFoster City, CA 94404GSAC/2000Applied Biosystems
AbstractThe ABI PRISM 3100 Genetic Analyzer is a 16–capillary electrophoresissystem allowing both sequencing and fragment analysis to be performed atmedium-to-high throughput. Its walk-away automation allows 24-hourunattended operation delivering a large reduction in labor costs over slab-gelbased systems. The system’s capabilities of running various lengths ofcapillaries and polymers provide endless possibilities for researchers to developa variety of applications with robust operation and high sensitivity.In this poster we will present performance evaluations of microsatellite andsingle nucleotide polymorphism (SNP) genotyping assays developed on the 3100analyzer. High performance microsatellite DNA sizing has been routinelyachieved with less than 0.15 nt standard deviation. In a 24 hour period, up to32 runs can be completed with total turnaround time less than 45 minutes usingTMa 36 cm array filled with POP-4 polymer. In addition four-color SNP analysiscan be obtained in less than 20 minutes with a 36 cm array and less than 12minutes with a 22 cm array. Examples of different genotyping assays forlinkage studies, forensic identification will be illustrated. Key features on the3100 analyzer will be covered as well.The ABI PRISM 3100 Genetic Analyzer includes patented technology licensed from Hitachi, Ltd. as part of astrategic partnership between Applied Biosystems and Hitachi, Ltd., as well as patented technology of AppliedBiosystems.Applied BiosystemsGSAC/2000
ABI PRISM 3100 Genetic Analyzer Front View 16 capillaries Fully automated with24 hours unattendedoperation Lower running andlabor cost providingmedium-to-highthroughput forsequencing andfragment analysisapplications PC running onWindows NT SoftwareGSAC/2000Applied Biosystems
ABI PRISM 3100 Genetic Analyzer: Doors OpenGSAC/2000Applied Biosystems
Instrument Open-Oven/Array/SampleTray Force air oven withtwo internal fans toprovide superiorthermal control anduniformity Array combs that clipinto oven to providereproducible arrayspatial alignment Accommodates two96- or 384- sampleplatesGSAC/2000Applied Biosystems
3100 System: With DifferentLengths of Capillary Arrays Uncoated capillariesfilled with POP-4 orPOP-6 polymer as asieving matrix Supports capillarylengths 36 cm and 50 cm Accommodates twoadditional capillarylengths 22 cm and 80 cm Loading header withintegrated capillary andelectrode Polymer block ferruleallows easy attachment ofall 16 capillaries to thepolymer blockTMTMGSAC/2000Applied Biosystems
Capillary Array:Detection Cell On-column detectionwith dual sideillumination to eliminatedifferential signalvariation and maximizethe sensitivity Supports more than 4dye detectionGSAC/2000Applied Biosystems
3100 System Workflow Easier and faster instrumentset-up Low running & labor cost Flexible run modules andconditions Variety of applicationsGSAC/2000Applied Biosystems
Fragment Analysis Kits CurrentlySupported on the 3100 System! Linkage Mapping SetHD-5! Linkage Mapping SetMD-10! Linkage Mapping Set LD20! Genescan 500 ROXSize Standard! Genescan 400HD ROXSize Standard! SNaPshot ddNTP PrimerExtension KitTMTMTMTMTMGSAC/2000! Genome screening at 5cM resolution! Genome screening at 10cM resolution! Genome screening at 20cM resolution! Size unknown fragments! Size unknown fragments! SNP analysis, screeningand validationApplied Biosystems
Spectral EXFAMApplied Biosystems
Fragment Analysis ModulesSupported on the 3100 SystemMODULERun TempCap Fill VolumePre-Run VoltagePre-Run TimeInjection VoltageInjection TimeRun VoltageData Delay TimeRun TimeSampling RateTurnaround TimeSpect36 POP4 GeneScan36 POP460 AC/200060 36 POP460 C184steps15kv16kv10s151s700s140ms28minsApplied Biosystems
Four-Color Microsatellite DNA Analysison the 3100 Genetic AnalyzerD9S288D9S1690TMElectropherogram of GeneScan Installation Standard sized withROX -labeled 400-HD size standard using 36 cm array filled withPOP-4 polymer. The Installation Standard is a pool of six linkageMapping Set markers, amplified from an individual CEPH familymember (1347-02) with FAM , HEX , NED dye-labeled primers.TMTMTMGSAC/2000TMTMApplied Biosystems
Cross Platform Comparison of HD5Linkage Mapping Set3773103100GSAC/2000Applied Biosystems
Relative Size AccuracyDYELOCIInstrument6-FAM D20S119Allele Allele12HEX D5S424Allele Allele12NED D9S288Allele plied Biosystems
Sizing PrecisionDYE6-FAM HEX NED LOCID20S119D5S424D9S288ALLELE SIZES113119217219138146ACROSS ARRAY0.05 0.04 0.06 0.06 0.05 0.04RUN to RUN0.04 0.04 0.07 0.07 0.05 0.06INSTRUMENT to0.09 0.08 0.13 0.13 0.08 0.09INSTRUMENTGSAC/2000Applied Biosystems
Microsatellite Analysis SummaryRelevant Features: Sensitivity Multicolor Decomponenting Resolution Relative Sizing Accuracy Sizing Precision ThroughputPerformanceNo dialysis requiredGoodSingle base resolution up to 400ntGood 0.15nt std dev32 runs/7,680 genotypes/24hr(with 15 GTs per capillary)GSAC/2000Applied Biosystems
SNP Genotyping Using 36 cm Arrayand POP-4 Polymer With E Dye SetTM29A45C34T38GFour pooled single nucleotide primer extension reactionproducts analyzed on the 3100 system using 36 cm array filledwith POP-4 polymer. The reaction was made from pGEM control DNA template provided by SNaPshot ddNTPPrimer Extension Kit and each product was labeled withdR110, dR6G, dTAMRA and dROX dye.Applied BiosystemsTMGSAC/2000
SNP Genotyping with 20 cm Array(Total Turnaround Time Less Than 12 Minutes)29A45C34TRun Condition:38GInjection: 2.5 KV, 10 sec.Run: 15 KV (484 V/cm), 60 C, POP4GSAC/2000Applied Biosystems
SNP Throughput (36 cm vs 22 cm)Array Type Turnaround Runs perTimeDaySamplesper DayGenotyping per Day(6 loci per injection)36cm20 min721,1526,91222cm12 min1201,92011,520GSAC/2000Applied Biosystems
Future Forensic Applicationon the 3100 Analyzer with F Dye SetElectropherogram of AmpFlSTR SGM Plus Allelic Laddersized with ROX dye-labeled 500 size standard using 36 cmarray filled with POP-4 polymer. The allelic ladder labeledwith 5-FAM , JOE and NED dyes was separatelydemonstrated in blue, green and black color.TMTMGSAC/2000Applied Biosystems
Fragile X Kit Analysis on the3100 System With C Dye SetHomozygoteHeterozygotePremutation AllelesElectropherogram of the control DNA from the fragile X sizepolymorphism assay kit sized with TAMRA dye-labeled 500size standard using 36 cm array filled with POP-4 polymer.Control DNA was labeled with TET and HEX dye.TMGSAC/2000Applied Biosystems
Applied Biosystems, 2001. All rights reserved.For Research Use Only. Not for use in diagnostic procedures.Applied Biosystems, ABI PRISM and its design, andAmpFLSTR are registered trademarks and 6-FAM,AB(Design),AmpFLSTR SGM Plus, Applera, FAM, ROX, TAMRA, FAM,GenBase, HEX, JOE, NED, POP-4, POP-6, ROX, SGM Plus,SnaPshot are trademarks of Applera Corporation or itssubsidiaries in the US and certain other countries.Windows NT is a registered trademark of MicrosoftCorporation. pGEM is a registered trademark of PromegaCorporation.GSAC/2000Applied Biosystems
The ABI PRISM 3100 Genetic Analyzer is a 16-capillary electrophoresis system allowing both sequencing and fragment analysis to be performed at medium-to-high throughput. Its walk-away automation allows 24-hour unattended operation delivering a large reduction in labor costs over slab-gel based systems. The system's capabilities of running .
