An integrated microfluidic device for DNA purification and PCR amplification of STR fragments
ABSTRACT This work presents the integration of DNA extraction from complex samples and PCR amplification of STR fragments in a valveless, glass microdevice, using commercially available kits and instrumentation. DNA extraction was performed using a microchannel packed with a silica solid phase and a standard syringe pump as a single pressure source driving the extraction process, followed by integrated, online microchip amplification of STR fragments in a total volume of 1.2 microL. Reported characteristics important to this work include the capacity of the device for purification of DNA from a complex biological sample (whole blood) and the timing of DNA elution from the silica solid phase for successful downstream PCR amplification by placement the microdevice into a conventional thermocycler. Potential application of this microdevice to forensic genetic analysis was demonstrated through the preliminary extraction of DNA from semen, followed by an integrated, multiplexed, on-chip amplification that yielded detectable STR amplicons. By utilizing conventional laboratory equipment, the device presented exploits the benefits of microfluidic systems without complex control systems.
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ABSTRACT: Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT(®) System, capable of producing up to five high quality STR profiles with full controls in approximately 90min using PowerPlex(®)16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis. The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform. are concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
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ABSTRACT: Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml(-1)), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.Biomicrofluidics 10/2014; 8(5):54119. DOI:10.1063/1.4899059 · 3.77 Impact Factor
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ABSTRACT: Microfluidic systems for polymerase chain reaction (PCR) should be fully closed to avoid vapor loss and to exclude the risk of contaminating the test environment. In closed systems however, the high temperatures of up to 95°C associated with PCR cause high overpressures up to 100 kPa, dominated by the increase of vapor partial pressure upon evaporation. Such high overpressures pose challenges to the mechanical stability of microfluidic chips as well as to the liquid handling in integrated sample-to-answer systems. In this work, we drastically reduce the pressure increase in fully closed PCR systems by integrating a microchannel that serves as a vapor-diffusion barrier (VDB), separating the liquid-filled PCR chamber from an auxiliary air chamber. In such configurations, propagation of vapor from the PCR chamber into the auxiliary air chamber and as a consequence the increase of pressure is limited by the slow diffusion process of vapor through the VDB. At temperature increase from 23°C to 95°C, we demonstrate the reduction of overpressure from more than 80 kPa without the VDB to only 35 kPa with the VDB. We further demonstrate proper function of VDB and its easy integration with downstream processes for PCR based nucleic acid amplification within centrifugal microfluidics. Without integration of the VDB, malfunction due to pressure-induced delamination of the microfluidic chip occurred.Lab on a Chip 12/2014; 15(4). DOI:10.1039/C4LC01115E · 5.75 Impact Factor