ABI PRISM 3100 Genetic Analyzer: A Fully Automated and High-Performance 16-Capillary Electrophoresis System for Fragment Analysis Applications

Transcription

1 ABI PRISM 3100 Genetic Analyzer: A Fully Automated and High-Performance 16-Capillary Electrophoresis System for Fragment Analysis Applications Yiwen Wang, Liwei Qi, Kitland Louie, Dong Wei, Xiaohui Chen, Ruben Pingue, Cydne Holt, Ben Johnson, Eric Nordman, Jing Tian, Ariana Wheaton, Nathan Caffo and Penny Dong 850 Lincoln Centre Drive Foster City, CA 94404

2 Abstract 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 various lengths of capillaries and polymers provide endless possibilities for researchers to develop a variety of applications with robust operation and high sensitivity. In this poster we will present performance evaluations of microsatellite and single nucleotide polymorphism (SNP) genotyping assays developed on the 3100 analyzer. High performance microsatellite DNA sizing has been routinely achieved with less than 0.15 nt standard deviation. In a 24 hour period, up to 32 runs can be completed with total turnaround time less than 45 minutes using a 36 cm array filled with POP-4 TM polymer. In addition four-color SNP analysis can be obtained in less than 20 minutes with a 36 cm array and less than 12 minutes with a 22 cm array. Examples of different genotyping assays for linkage studies, forensic identification will be illustrated. Key features on the 3100 analyzer will be covered as well. The ABI PRISM 3100 Genetic Analyzer includes patented technology licensed from Hitachi, Ltd. as part of a strategic partnership between and Hitachi, Ltd., as well as patented technology of Applied Biosystems.

3 ABI PRISM 3100 Genetic Analyzer Front View 16 capillaries Fully automated with 24 hours unattended operation Lower running and labor cost providing medium-to-high throughput for sequencing and fragment analysis applications PC running on Windows NT Software

4 ABI PRISM 3100 Genetic Analyzer: Doors Open

5 Instrument Open-Oven/Array/SampleTray Force air oven with two internal fans to provide superior thermal control and uniformity Array combs that clip into oven to provide reproducible array spatial alignment Accommodates two 96- or 384- sample plates

6 3100 System: With Different Lengths of Capillary Arrays Uncoated capillaries filled with POP-4 TM or POP-6 TM polymer as a sieving matrix Supports capillary lengths 36 cm and 50 cm Accommodates two additional capillary lengths 22 cm and 80 cm Loading header with integrated capillary and electrode Polymer block ferrule allows easy attachment of all 16 capillaries to the polymer block

7 Capillary Array:Detection Cell On-column detection with dual side illumination to eliminate differential signal variation and maximize the sensitivity Supports more than 4 dye detection

8 3100 System Workflow Easier and faster instrument set-up Low running & labor cost Flexible run modules and conditions Variety of applications

9 Fragment Analysis Kits Currently Supported on the 3100 System! Linkage Mapping Set HD-5! Linkage Mapping Set MD-10! Linkage Mapping Set LD- 20! Genescan TM 500 ROX TM Size Standard! Genescan TM 400HD ROX TM Size Standard! SNaPshot TM ddntp Primer Extension Kit! Genome screening at ~5cM resolution! Genome screening at ~10cM resolution! Genome screening at ~20cM resolution! Size unknown fragments! Size unknown fragments! SNP analysis, screening and validation

10 Spectral Calibration Before After ROX NED HEX FAM ROX NED HEX FAM

11 Fragment Analysis Modules Supported on the 3100 System MODULE Spect36 POP4 GeneScan36_POP4 SNP36_POP4 Run Temp 60 C 60 C 60 C Cap Fill Volume 184steps 184steps 184steps Pre-Run Voltage 15kv 15kv 15kv Pre-Run Time 180s 180s 1 Injection Voltage 1.5kv 7.5kv 6kv Injection Time 10s 10s 10s Run Voltage 15kv 15kv 15 Data Delay Time 100s 1s 1s Run Time 800s 1500s 700s Sampling Rate 140ms 140ms 140ms Turnaround Time 30mins 45mins 28mins

12 Four-Color Microsatellite DNA Analysis on the 3100 Genetic Analyzer D9S288 D9S1690 Electropherogram of GeneScan TM Installation Standard sized with ROX TM -labeled 400-HD size standard using 36 cm array filled with POP-4 TM polymer. The Installation Standard is a pool of six linkage Mapping Set markers, amplified from an individual CEPH family member ( ) with FAM TM, HEX TM, NED TM dye-labeled primers.

13 Cross Platform Comparison of HD5 Linkage Mapping Set

14 Relative Size Accuracy DYE 6-FAM HEX NED LOCI D20S119 D5S424 D9S288 Instrument Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele /POP /POP /POP /POP

15 Sizing Precision DYE 6-FAM HEX NED LOCI D20S119 D5S424 D9S288 ALLELE SIZES ACROSS ARRAY RUN to RUN INSTRUMENT to INSTRUMENT

16 Microsatellite Analysis Summary Relevant Features: Sensitivity Multicolor Decomponenting Resolution Relative Sizing Accuracy Sizing Precision Throughput Performance No dialysis required Good Single base resolution up to 400nt Good <0.15nt std dev 32 runs/7,680 genotypes/24hr (with 15 GTs per capillary)

17 SNP Genotyping Using 36 cm Array and POP-4 TM Polymer With E Dye Set 29 A 34 T 38 G 45 C Four pooled single nucleotide primer extension reaction products analyzed on the 3100 system using 36 cm array filled with POP-4 polymer. The reaction was made from pgem control DNA template provided by SNaPshot TM ddntp Primer Extension Kit and each product was labeled with dr110, dr6g, dtamra and drox dye.

18 SNP Genotyping with 20 cm Array (Total Turnaround Time Less Than 12 Minutes) 29 A 34 T 38 G 45 C Run Condition: Injection: 2.5 KV, 10 sec. Run: 15 KV (484 V/cm), 60 C, POP4

19 SNP Throughput (36 cm vs 22 cm) Array Type Turnaround Time Runs per Day Samples per Day Genotyping per Day (6 loci per injection) 36cm 20 min 72 1,152 6,912 22cm 12 min 120 1,920 11,520

20 Future Forensic Application on the 3100 Analyzer with F Dye Set Electropherogram of AmpFlSTR SGM Plus TM Allelic Ladder sized with ROX dye-labeled 500 size standard using 36 cm array filled with POP-4 TM polymer. The allelic ladder labeled with 5-FAM, JOE and NED dyes was separately demonstrated in blue, green and black color.

21 Fragile X Kit Analysis on the 3100 System With C Dye Set Homozygote Heterozygote Premutation Alleles Electropherogram of the control DNA from the fragile X size polymorphism assay kit sized with TAMRA dye-labeled 500 size standard using 36 cm array filled with POP-4 TM polymer. Control DNA was labeled with TET and HEX dye.

22 , All rights reserved. For Research Use Only. Not for use in diagnostic procedures., ABI PRISM and its design, and AmpFLSTR 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 its subsidiaries in the US and certain other countries. Windows NT is a registered trademark of Microsoft Corporation. pgem is a registered trademark of Promega Corporation.