Robert Blazej

High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets.

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.

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.

MICROFABRICATED CAPILLARY ARRAY ELECTROPHORESIS: HIGH-THROUGHPUT DNA SEQUENCING AND POLYMORPHISM ANALYSIS

Microfabricated capillary array electrophoresis (µCAE) circuitry has been developed and optimized allowing high-throughput DNA sequencing and polymorphism analysis in a compact, circular, wafer-scale device. The 96-lane processor, which incorporates hyper-turns and fluidically balanced injectors, produces ∼41,000 bases of M13 extension products to an accuracy >99% (phred 20) in only 24 minutes. Polymorphism ratio sequencing (PRS) with the same device enables rapid scanning of the entire human mitochondrial genome in a single run.

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.