Lance Koutny

Immunoassay for thyroxine (T4) in serum using capillary electrophoresis and micromachined devices

As part of our ongoing effort to develop electrophoretic assay technology for clinical diagnostics, we describe a competitive immunoassay for the determination of serum thyroxine (T4) based on electrophoresis and laser induced fluorescence (LIF). Measurements of total T4 are useful for the clinical evaluation of thyroid function. A fluorescein thyroxine conjugate was utilized in conjunction with a polyclonal antibody preparation as assay reagents. Capillary electrophoresis (CE) conditions tolerant of the direct injection of serum without extraction or other sample preparation steps were developed and used for quantitation of total T4 in serum. We have been exploring the use of micromachined devices with arrays of channels for high assay throughput. Our assay protocol was carried in a microchip format. The results illustrate that gains in speed can be additionally achieved, with the electrophoretic separation of free from bound labelled T4 being performed in about 15 s for serum samples.

High-speed analysis of multiplexed short tandem repeats with an electrophoretic microdevice

We report the development of a robust and effective method for multiplexed short tandem repeat (STR) analysis within a chip‐based microdevice. The method uses a laser‐induced fluorescence detection system and simultaneously detects three‐ and four‐color multiplexed polymerase chain reaction (PCR) samples. Analyses of the eight combined DNA index system (CODIS) STR loci were performed in 20 min with single‐base‐pair resolution ranging from 0.75 to 1. A simultaneous analysis of fifteen loci‐ladders and a gender marker Amelogenin based on the PowerPlex™ 16 System was achieved in less than 35 min. The system is capable of repetitive operation and may be extended to high‐throughput multilane devices that could be readily interfaced to an automated sample loading system.

Recent advances in DNA sequencing by capillary and microdevice electrophoresis

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highlight the recent progress in aspects of capillary electrophoresis (CE) that has enabled these achievements. In addition, we summarize recent developments in the use of microfabricated devices for DNA sequencing that promise to bring the next leap in productivity.

Recent developments in DNA sequencing by capillary and microdevice electrophoresis

The present review covers papers published in the years 1997 and 1998 on DNA sequencing by capillary and microdevice electrophoresis. The article does not include other electrophoretic DNA applications such as analysis of oligonucleotides, genotyping, and mutational analysis. Capillary gel electrophoresis (CGE) is starting to become a viable competitor to slab gel electrophoresis for DNA sequencing. Commercially available multicapillary array sequencers are now entering sequencing facilities which to date have totally relied on traditional slab gel technology. CGE research on DNA sequencing therefore becomes increasingly concerned with the critical task of fine‐tuning the operational parameters to create robust sequencing systems. Electrophoretic microdevices are being considered the next technological step in DNA sequencing by electrophoresis.

Automated image analysis for distortion compensation in sequencing gel electrophoresis

A computerized method is described for correcting systematic distortion of images from slab gel electrophoresis. Such distortions can lead to misinterpretation of the information contained in the gel. The method is useful for data analysis in one-dimension slab gel electrophoresis where the information is manifested in rectangular shaped bands, such as conventional restriction digest or sequencing gels, and the distortions can be adequately described by continuous low-order polynomial functions. The purpose is to eliminate human skill and judgement from the process and to minimize human interaction, which would be useful in any future attempts to automate the analysis of electrophoretic gels.

MEMS-based systems for DNA sequencing and forensics

We outline some of the most important scientific problems that were overcome in this multiyear project. These include (a) the high-yield microfabrication of zero-defect bonded glass plates at sizes greatly exceeding semiconductor fabrication, (b) the integration of electrodes, reservoirs, and high-pressure seals into these plates, (c) detector optimization for the required very high rate data acquisition and (d) microfluidics issues and optimizations related to channel geometry.

BIOMEMS-768 DNA Sequencer

The Whitehead Institute has developed an automated DNA sequencer that will go into final Genome Center testing during the summer of 2001. The system comprises a total of 768 separation channels distributed over two plates. The working elements are 50-cm × 25-cm, 384-lane microfabricated glass elements, which undergo alternating electrophoresis and regeneration under use with an exchangeable sieving matrix. The microfluidic and sample transfer devices needed to service the plates are integrated into the same compact instrument. Concurrently with the instrument development we have developed all the protocols, materials and surface preparations to achieve matrix-limited separations of single-stranded DNA in the microfabricated plate format.