title: Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.
abstract: Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.
authors.affiliation: Department of Chemistry, University of Hull, Cottingham Road, Hull, UK HU6 7RX.
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: Shaw
authors.forename: Domino A
authors.initials: DA
authors.lastname: Joyce
authors.forename: Peter T
authors.initials: PT
authors.lastname: Docker
authors.forename: Charlotte E
authors.initials: CE
authors.lastname: Dyer
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Greenway
authors.forename: John
authors.initials: J
authors.lastname: Greenman
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Haswell
mesh_terms: D053523:Amelogenin / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods; D005782:Gels / Q000737:chemistry; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D011108:Polymers; D012822:Silicon Dioxide / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods*
chemical_list: D053523:Amelogenin; D005782:Gels; D011108:Polymers; D012822:Silicon Dioxide; D004247:DNA