title: Pyrosequencing in a microfluidic flow-through device.
abstract: To explore genome variation meaningfully, there is a critical need for a high-throughput and inexpensive platform for DNA analysis. Pyrosequencing is a nonelectrophoretic bioluminometric DNA sequencing method that uses a four-enzyme mixture reaction to monitor nucleotide incorporation in real time. Currently, the commercialized pyrosequencing technique is limited to a 96-microtiter plate format. However, high throughput and inexpensive pyrosequencing is required to meet the need of screening large numbers of samples. We present here DNA pyrosequencing on a nanoliter-volume microfluidic platform. The microfluidic approach involves the trapping of the DNA on microbeads in an on-chip filter chamber and flow-through of the pyrosequencing reagents to monitor the reaction in real time. Two single-nucleotide polymorphisms were successfully scored to evaluate the microfluidic platform. In addition to significantly reducing reagent costs, microfluidic systems promise to improve the read length by eliminating intermediate product accumulation by constant removal of unincorporated nucleotides and elimination of dilution effects at each reaction cycle in the current plate format. Although only one filter chamber was used in this study, the platform should be readily adaptable to parallel analyses of nanoliter samples using filter chamber arrays to obtain high-throughput DNA analysis.
authors.affiliation: Department of Signals, Sensors and Systems, Microsystem Technology, Royal Institute of Technology, Stockholm, Sweden. arusson@partners.org
authors.forename: Aman
authors.initials: A
authors.lastname: Russom
authors.forename: Nigel
authors.initials: N
authors.lastname: Tooke
authors.forename: Helene
authors.initials: H
authors.lastname: Andersson
authors.forename: Göran
authors.initials: G
authors.lastname: Stemme
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry; D044085:Microfluidics / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D009711:Nucleotides / Q000737:chemistry
chemical_list: D009711:Nucleotides; D004247:DNA