White Paper: High Throughput SNP Genotyping Using Array Tape in Place of Microplates

Transcription

1 White Paper High Throughput SNP Genotyping White Paper: High Throughput SNP Genotyping Using Array Tape in Place of Microplates Miniaturization and Automation Using a Novel New Reaction Substrate Originally published in January Reformatted document in Seth Dobrin 1, Donna David 1, Kim Fieweger 1, Bill Dickinson 1, Lauri Ott 1, Terry Rusch 1, Jon Chudyk 1, James Weber 2, and Elaine May 3 1 Center for Human Genetics, Marshfield Clinic Research Foundation, Marshfield, WI 2 Prevention Genetics, Marshfield, WI 3 Global Array, Minneapolis, MN (merged with Douglas Scientific in 2009)

2 White Paper High Throughput SNP Genotyping 1 Overview Increased throughput requirements for SNP genotyping have led to many high throughput technologies such as microarrays and gene chips that forgo traditional aqueous phase genotyping methods. The use of array tape in place of microplates provides an automated, miniaturized format which maintains the flexibility and accuracy of aqueous phase genotyping assays. Array tape (also known as Microtape ) is a continuous plastic tape in which standard SBS format well patterns are sequentially embossed, allowing processing of the equivalent of hundreds of microplates on one reel of tape. Advantages of this technology include streamlining of automation, and with miniaturized well sizes, cost savings of up to 90% on reagents and consumables. This format also allows for on-reel simultaneous waterbath thermal cycling of hundreds of microplate equivalents at one time, and is therefore ideal for PCR based processes. Array Tape (now available from Douglas Scientific*) At the Center for Human Genetics at the Marshfield Research foundation, and also at Prevention Genetics, both in Marshfield, Wisconsin, array tape based high throughput SNP genotyping has been implemented very successfully for many assay types, including allele specific PCR assays (such as molecular beacon assays) and TaqMan assays with results equal to those obtained on microplates. We have found array tape technology to be the method of choice for fine mapping regions of interest in whole genome linkage scans or whole genome association scans. Materials/Methods Array Tape and Cover Tape Reactions were carried out in 384-well polypropylene Microtape, currently available from Douglas Scientific*. Wells were sealed with an optically clear, low autofluorescent polyolefin cover tape, also available from Douglas Scientific*. Liquid Handling and Tape Sealing Equipment DNA samples were transferred into tape using a 384 tip positive displacement pipetting head. Common reagent solution was added using a single head non-contact

3 White Paper High Throughput SNP Genotyping 2 solenoid valve dispensor. Original equipment was developed at the Marshfield Clinic, and is now available in a commercialized version manufactured by Douglas Scientific* (see below). The Nexar This configuration includes pipetting, drying, dispensing, and cover tape sealing modules. Waterbath Thermal Cycler Multiple arrays were simultaneouslly thermal cycled in by sequential dipping in a waterbath thermal cycler developed in-house. Detection Equipment and Software Detection equipment was developed at the Marshfield Clinic and Prevention Genetics, along with image processing and scoring software. Our ArrayProcessor program processes the raw image data, and Plate Scorer uses this data to render images of the raw or separataed marker dye channels and display scatter plots of the well intensities. Clusters are manually scored by enclosing them in polygons and assigning scores using a context menu displayed over the cluster by a right mouse button click. (Douglas Scientific* is also introducing commercially available detection units.) Allele-Specific PCR Assay Protocol 5ng of template DNA in 800nl of 10mM Tris, 0.2mM EDTA, ph of 8.0 is pipetted into the 384 well array tape and dried. 800 nl of a common reagent solution containing 1X PCR buffer, 600nM of common primer, 45 nm each Allele-specific primer, 2.5mM Mg+2, 37.5 nm Fam UniPrimer, 50 nm Joe UniPrimer (Chemicon), 250μM dntps, 1% Rox reference dye solution (Invitrogen, Cat. No ), and 0.05 units/μl Platinum Taq polymerase (Invitrogen) is then added to the entire array. A sealing cover tape is then applied. The samples are then amplified in a waterbath thermal cycler under the following conditions: 95 C x 2 minutes, followed by 35 cycles of 95 C x 20 seconds, 55 C x 40 seconds, 72 C x 20 seconds, followed by a final extension at 72 C x 6 minutes. TaqMan Assay Protocol The TaqMan protocol is basically similar to the above allele-specific PCR protocol, with the following variations: 5ng of template DNA is pipetted into the plate and dried. 800 nl of a reaction mixture containing 1 X PCR Buffer, 100 μm dntps, 0.05 units/μl Platinum Taq, 1.5 mm Mg+2, 1% Rox reference dye solution (Invitrogen, Cat. No ), and 0.15 X TaqMan primers and probes (primers: μm, probes: μm) (Applied Biosystems) is then added to the dried DNA in the tape. The samples are amplified in a waterbath thermal cycler with the following cycle pattern: 95 C for 3 minutes, then 35 cycles of 95 C x 20 seconds, 60 C x 1 minute, with a final incubation at 60 C for 6 minutes.

4 White Paper High Throughput SNP Genotyping 3 3 Results Assay Design Considerations: Percent Failure Rates by Assay Type Once assays have been designed and validated, overall completion rates approach 99% with good quality DNA. 0 Allele-Specific PCR Custom TaqMan Pre-validated TaqMan Representative Scatter Plot of TaqMan Assay on Array Tape Representative Scatter Plot of Allele-Specific PCR Assay on Array Tape Project Number of Genotypes Error Rate (%) 1 94, , , , , , , TOTAL 547, Error Rates by Project

5 White Paper High Throughput SNP Genotyping 4 Results Genotypes per Technician per Shift in Array Tape vs. Microplates Reaction Volume in Microliters for TaqMan Assay in Array Tape vs. Microplates Discussion Of the two chemistries described here, TaqMan is the main one that we use, and we primarily use it for fine mapping. The TaqMan assay usually has more locus specifity than allele-specific PCR because not only must both PCR primers anneal, but both probes must anneal to the correct site. This factor greatly reduces background signal in the reaction. Because of this increased locus specificity, the TaqMan assay is also well-suited to working with gene families and other repeated sequences. The primary drawbacks to the Taqman assay are that synthesis of a specific pair of dual-labeled probes must be carried out for each polymorphism, and there is an inability to multiplex. TaqMan assays typically require a 25 μl reaction volume. Our current reactions are carried out at 700nl, a 15-fold reduction in volume and in cost of reagents. While volumes down to 300nl are clearly viable, we act conservatively and use a higher volume to ensure high quality calls.

6 White Paper High Throughput SNP Genotyping 5 Continued reduction in volume will further drive down cost and conserve highly valuable patient DNA samples. Similarly, multiplexing assays is being explored as a means to further reduce costs and conserve DNA. Conclusion Array tape technology is an excellent method for cost-effective, high throughput SNP genotyping for fine mapping regions of interest in whole genome linkage scans or whole genome association scans. Until recently, the use of array tape has been limited to use in the Marshfield Clinic and at Prevention Genetics because all instrumentation was developed in-house. The introduction of modular commercialized array tape equipment by Douglas Scientific* will make this method adaptable to any type of assay currently being done in microplates, including DNA sequencing, STRP genotyping, and pharmaceutical assays. The Douglas Scientific Modular Array Tape Unit Modules are interchangeable and easily re-configured for different processes and applications. The Marshfield Clinic Tel: info@douglasscientific.com *Douglas Scientific merged with Global Array in 2009.