Biomedical subjects

Robert G Blazej

Publications and source records attributed to Robert G Blazej.

3 recordsLinked to original sources

PubMed2006

Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing.

An efficient, nanoliter-scale microfabricated bioprocessor integrating all three Sanger sequencing steps, thermal cycling, sample purification, and capillary electrophoresis, has been developed and evaluated. Hybrid glass-polydimethylsiloxane (PDMS) wafer-scale construction is used to combine 250-nl reactors, affinity-capture purification chambers, high-performance capillary electrophoresis channels, and pneumatic valves and pumps onto a single microfabricated device. Lab-on-a-chip-level integration enables complete Sanger sequencing from only 1 fmol of DNA template. Up to 556 continuous bases were sequenced with 99% accuracy, demonstrating read lengths required for de novo sequencing of human and other complex genomes. The performance of this miniaturized DNA sequencer provides a benchmark for predicting the ultimate cost and efficiency limits of Sanger sequencing.

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PubMed2003

Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis.

Modern DNA sequencing 'factories' have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a 'sequencing lab-on-a-chip'. Integrated microfluidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.

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PubMed2003

Polymorphism ratio sequencing: a new approach for single nucleotide polymorphism discovery and genotyping.

Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer.

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