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.

A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-μL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-μL human whole blood followed by amplifications of 59-bp β-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

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We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-ll pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, "amplicon-in-answer-out" operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis. V C 2015 AIP Publishing LLC.