SNP Genotyping on Array Tape
PreventionGenetics has converted all of its SNP and insertion-deletion polymorphism genotyping, previously performed in low-volume, 384-well PCR microplates, to processes performed in Global Array 384-well polypropylene 1.5-µL array tape. This has involved reducing the reaction volume from 5 µL to about 800 nL. It was found that for both allele-specific and TaqMan assays, using the same concentrations of reagents as in plates in lower volumes in tape yields good results. While reaction volumes of as little as 300 nL have yielded scorable genotypes, an 800-nL reaction volume yields more consistent results. The final reaction protocol for allele-specific PCR assays in array tape is as follows: 5 ng 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 1 X PCR buffer, 600 nM of common primer, 45 nM each of allele-specific primer, 2.5mM Mg+2, 37.5 nM Fam UniPrimer, 50 nM Joe UniPrimer (Chemicon), 250 µM dNTPs, 1% Rox reference dye solution (Invitrogen), and 0.05 units/µL Platinum Taq® polymerase (Invitrogen) is then added to the entire array. A sealing cover tape is applied. The samples are amplified in a water bath 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.
The TaqMan protocol is basically similar to the above allele-specific PCR protocol, with the following variations: 5 ng 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), and 0.15 X TaqMan primers and probes (primers: 0.134 µM, probes: 0.030 µM) is then added to the dried DNA in the tape. The samples are amplified in a water bath 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.
The prethermal cycling processes of pipetting and drying template DNA, dispensing reaction mixture, and applying a sealing cover tape are performed in a continuous process on sequential Douglas Scientific modules. The template DNA is pipetted from sample plates into tape using a 384-tip syringe type pipettor (Roche).
The samples are then passed over drying modules where all liquid is evaporated from the DNA samples. A Douglas Scientific jet-type dispenser delivers the reaction mixture. A low autofluorescence polyolefin clear Global Array cover tape is then applied. The sealed arrays are transferred to a water bath thermal cycler for amplification. Following thermal cycling, arrays are transferred to a detection module, and detection is performed through the cover tape.
PreventionGenetics has developed its own fluorescence readers that feed tape in a continuous fashion. The raw data are processed and scored with software developed in-house. A commercial detection unit is available through Douglas Scientific.