Charles A. Emrich

Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis

A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 degree C s(-1). The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (μCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q ≥ 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The μCAE system demonstrated here produces sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of μCAE technology should make it the next generation, high performance DNA sequencing platform.

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates

This review focuses on some recent advances in realizing microfabricated capillary array electrophoresis (νCAE). In particular, the development of a novel rotary scanning confocal fluorescence detector has facilitated the high‐speed collection of sequencing and genotyping data from radially formatted νCAE devices. The concomitant development of a convenient energy‐transfer cassette labeling chemistry allows sensitive multicolor labeling of any DNA genotyping or sequencing analyte. High‐performance hereditary haemochromatosis and short tandem repeat genotyping assays are demonstrated on these devices along with rapid mitochondrial DNA sequence polymorphism analysis. Progress in supporting technology such as robotic fluid dispensing and batched data analysis is also presented. The ultimate goal is to develop a parallel analysis platform capable of integrated sample preparation and automated electrophoretic analysis with a throughput 10–100 times that of current technology.

Use of the PURExpress® In Vitro Protein Synthesis Kit, Disulfide Bond Enhancer and SHuffle™ Competent E. coli for Heterologous In Vitro and In Vivo Cellulase Expression

Advances in industry and medicine have led to the engineering of complex “designer” proteins, such as antibodies in targeted therapeutics and enzymes in process development. The ability to easily generate an almost infinite number of variants at the DNA level has increased the demand for improved protein expression methodologies to fully capture what can be produced genetically. Often, the protein of interest is eukaryotic in origin and may require posttranslational modifications specific to its native host or may be toxic to the host cells expressing them. Cell-free protein expression systems have allowed us to step beyond the limits of traditional in vivo expression methodologies by decoupling protein expression from host cell viability (1,2,3). Furthermore, the ability to produce complex proteins using cell-free transcription/translation systems uniquely enables high-throughput directed evolution and protein engineering efforts (4,5). Several cell-free protein expression systems have been developed in the last decade with recent advances focusing on special folding or assembly environments (6,7,8). Equally as important is the capability to transition from the in vitro system to largerscale in vivo expression, while maintaining activity of the target protein (9,10).

Microfabricated capillary array electrophoresis: Implementation and applications

Publisher Summary This chapter outlines the progress toward novel microfabricated capillary array electrophoresis (μCAE)) devices capable of fully integrated, high-throughput genotyping and DNA sequencing. These integrated bio processors lead the way to the next generation of DNA analysis devices. Initial capillary array electrophoresis (CAE) efforts utilized bundles of drawn, fused-silica capillaries that provide significant advantages in analysis time, sample volume, and process automation over conventional slab gels. A modified design was presented in which 12 capillaries were bundled into a ∼1.1 mm region to facilitate scanned sample interrogation. The ability to fabricate dense array devices reproducibly in an industry-standard wafer format facilitates the continued evolution toward integrated bio processors containing not only arrays of high-performance DNA separation columns, but also thermal-cycling reaction and sample purification chambers. Continued progress on this trajectory can yield devices capable of performing the most sophisticated genetic assays in a completely integrated format.