title: Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.
abstract: The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.
authors.affiliation: Cepheid, Sunnyvale, CA 94089, USA.
authors.forename: Fariba
authors.initials: F
authors.lastname: Raisi
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Blizard
authors.forename: Akbar
authors.initials: A
authors.lastname: Raissi Shabari
authors.forename: Jesus
authors.initials: J
authors.lastname: Ching
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kintz
authors.forename: Jim
authors.initials: J
authors.lastname: Mitchell
authors.forename: Asuncion
authors.initials: A
authors.lastname: Lemoff
authors.forename: Mike T
authors.initials: MT
authors.lastname: Taylor
authors.forename: Fred
authors.initials: F
authors.lastname: Weir
authors.forename: Linda
authors.initials: L
authors.lastname: Western
authors.forename: Wendy
authors.initials: W
authors.lastname: Wong
authors.forename: Rekha
authors.initials: R
authors.lastname: Joshi
authors.forename: Pamela
authors.initials: P
authors.lastname: Howland
authors.forename: Avinash
authors.initials: A
authors.lastname: Chauhan
authors.forename: Peter
authors.initials: P
authors.lastname: Nguyen
authors.forename: Kurt E
authors.initials: KE
authors.lastname: Petersen
mesh_terms: D001331:Automation; D001483:Base Sequence; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D015894:Genome, Human; D006801:Humans; D008904:Miniaturization / Q000379:methods; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004247:DNA