Complete High Throughput Pharmacogenomics Workflow: From Sample to Results

Application Note

Michael Farrell¹,Jeff Roeder¹, Travis Butts¹, Julie Baggs¹, Luke Linz², Mike Freeman², Steven Nesbit³, Joan Gordon³

¹Omega Bio-tek, Inc., ²Douglas Scientific., ³Maine Molecular Quality Controls, Inc.

Introduction

Rapid, high throughput, cost-effective and accurate screening of human DNA for the identification of single nucleotide polymorphisms (SNPs) is a useful tool for the development of therapeutics and diagnostics. Here, we demonstrate these metrics with an automated pharmacogenomics workflow consisting of a non-invasive buccal swab collection system and DNA extraction chemistry (Omega Bio-tek) on 2 open automated liquid handling platforms (Hamilton’s Microlab® STAR™ and Qiagen’s BioSprint® 96), followed by specific SNP identification via a high throughput genotyping instrument (Douglas Scientific). For demonstration purposes, this system was used to determine the SNP status of warfarin anticoagulant sensitivity mutations within the human VKORC1 and CYP2C9 genes (Ref 1). Genomic controls (Introl HD Multiplex WT, HET, and MUT) were provided by Maine Molecular Quality Controls, Inc.

Workflows and Methods

Omega Bio-tek’s E-Z 96® Spin-Out buccal swab collection device allows for the rapid arraying of sample swabs (1) into 96-well plates for lysis configuration (2), followed by the raised configuration (3) for easy eluate epin-out and start of automated workflow.

Omega Bio-tek’s Mag-BIND® Blood & Tissue DNA HDQ 96 Kit is designed for the rapid and reliable isolation of quality genomic DNA from swabs and other sample types. Mag-BIND® Particles HDQ provide quick magnetic response time. Utilizing paramagnetic particles allows use on multiple open-ended liquid handling platforms and provides high-quality DNA that is suitable for direct use in most downstream applications.

The IntelliQube® by Douglas Scientific combines liquid handling with real-time quantitative PCR (qPCR) analysis in miniaturized reaction volumes. The system utilizes Array Tape® in a unique and innovative 768-well format. Array Tape® is a thin and flexible polypropylene consumable that, in combination with miniaturized reaction volumes (1.6 μL) enables both outstanding PCR performance and profound reagent savings.

Results & Discussion

The purified DNA from 17 replicate samples was quantified for DNA concentration and total DNA yields via PicoGreen® (Figure 1). DNA from these samples was then analyzed via BHQplus® probe-based qPCR for wild-type, mutant, or heterozygous status for warfarin sensitivity at 3 known SNPs (Figures 2 & 3).

Figure 2. Genotype scatter plots from the IntelliQube®. WT = wild-type, MUT = mutant, HET = heterozygous.

Conclusions

These sample collection, extraction and analysis technologies offer a rapid, high throughput, low cost, and accurate pharmacogenomics workflow solution for human DNA genotyping.
The Douglas Scientific IntelliQube®, Omega Bio-tek’s Mag-BIND® Blood & Tissue DNA HDQ 96 Kit and Maine Molecular Quality Controls, Inc.’s products are for research only.

References

1. Takeuchi, F., et al. (2009). PLoS Genet 5: e1000433. doi: 10.1371.

Application Note

Introduction

Workflows and Methods

Results & Discussion

Conclusions

References

WP-0027