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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. [ABSTRACT FROM AUTHOR]

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 μL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25–1 μL of human whole blood yielded 8.1–21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 μm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, “in situ” PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems. [ABSTRACT FROM AUTHOR]

We have successfully developed an integrated microsystem that combines two plastic microchips for DNA extraction and PCR amplification with a glass capillary array electrophoresis chip together in a compact control and detection instrument for automated forensic short tandem repeat (STR) analysis. DNA extraction followed by an “in situ PCR” was conducted in a single reaction chamber of the microchip based on a filter paper-based extraction methodology. PCR products were then mixed with sizing standards by an injection electrode and injected into the electrophoresis chip for four-color confocal fluorescence detection. The entire STR analysis can be completed in about two hours without any human intervention. Since the 15-plex STR system has a more stringent requirement for PCR efficiency, we optimized the structure of the plastic DNA extraction and amplification chip, in which the reaction chamber was formed by sandwiching a hollow structure layer with two blank cover layers, to reduce the adsorption of PCR reagents to the surfaces. In addition, PCR additives, bovine serum albumin, poly(ethylene glycol), and more magnesium chloride were included into the on-chip multiplex STR system. The limit-of-detection study demonstrated that our microsystem was able to produce full 15-plex STR profiles from 3.75 ng standard K562 DNA. Buccal swab and whole blood samples were also successfully typed by our system, validating the feasibility of performing rapid DNA typing in a “sample-in-answer-out” manner for on-site forensic human identification. [ABSTRACT FROM AUTHOR]