A fully integrated and automated microsystem for rapid pharmacogenetic typing of multiple warfarin-related single-nucleotide polymorphisms

Abstract

A fully integrated and automated microsystem consisting of low-cost, disposable plastic chips for DNA extraction and PCR amplification combined with a reusable glass capillary array electrophoresis chip in a modular-based format was successfully developed for warfarin pharmacogenetic testing. DNA extraction was performed by adopting a filter paper-based method, followed by “in situ” PCR that was carried out directly in the same reaction chamber of the chip without elution. PCR products were then co-injected with sizing standards into separation channels for detection using a novel injection electrode. The entire process was automatically conducted on a custom-made compact control and detection instrument. The limit of detection of the microsystem for the singleplex amplification of amelogenin was determined to be 0.625 ng of standard K562 DNA and 0.3 μL of human whole blood. A two-color multiplex allele-specific PCR assay for detecting the warfarin-related single-nucleotide polymorphisms (SNPs) 6853 (–1639G>A) and 6484 (1173C>T) in the VKORC1 gene and the *3 SNP (1075A>C) in the CYP2C9 gene was developed and used for validation studies. The fully automated genetic analysis was completed in two hours with a minimum requirement of 0.5 μL of input blood. Samples from patients with different genotypes were all accurately analyzed. In addition, both dried bloodstains and oral swabs were successfully processed by the microsystem with a simple modification to the DNA extraction and amplification chip. The successful development and operation of this microsystem establish the feasibility of rapid warfarin pharmacogenetic testing in routine clinical practice.