Highly Parallelized Screening of Functionally Enhanced XNA Aptamers in Uniform Hydrogel Particles.

Xeno-nucleic acid (XNA) aptamers based on evolvable non-natural genetic polymers hold enormous potential as future diagnostic and therapeutic agents. However, time-consuming and costly procedures requiring the purification of individual XNA sequences produced by large-scale polymerase-mediated primer extension reactions pose a major bottleneck to the discovery of highly active XNA motifs for biomedical applications. Here, we describe a straightforward approach for rapidly surveying the binding properties of XNA aptamers identified by in vitro selection. Our strategy involves preparing XNA aptamer particles in which many copies of the same aptamer sequence are distributed throughout the gel matrix of a polyacrylamide-encapsulated magnetic particle. Aptamer particles are then screened by flow cytometry to assess target binding affinity and deduce structure-activity relationships. This generalizable and highly parallel assay dramatically accelerates the pace of secondary screening by allowing a single researcher to evaluate 48-96 sequences per day.

High-pressure gel loader for capillary array electrophoresis microchannel plates.

Microfabricated capillary array electrophoresis (microCAE) microchannel plates are the next generation of bioanalytical separation devices. To fully exploit the capabilities of microCAE devices, supporting technology such as robotic sample loading, gel loading, microplate washing, and data analysis must be developed. Here, we describe a device for loading gel into radial capillary array electrophoresis microplates and for plate washing and drying. The microplates are locked into a loading module, and high-pressure helium is used to drive aqueous separation media or wash solutions into the microchannels through fixtures connected to the central anode reservoir. Microplates are rapidly (30 s to 5 min) loaded with separation media, such as 3%-4.8% linear polyacrylamide or 0.7%-3.0% hydroxyethyl cellulose, for electrophoresis. The effective and rapid gel-filling and plate-cleaning methods together with short electrophoretic analysis times (2-30 min) make microCAE systems versatile and powerful nucleic acid analysis platforms.

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates.

This review focuses on some recent advances in realizing microfabricated capillary array electrophoresis (microCAE). 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 microCAE 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 DNA analysis systems.

Microfabricated "laboratory-on-a-chip" systems are revolutionizing all aspects of genetic analysis. The development of capillary array electrophoresis (CAE) microchannel plate devices makes possible the performance of 96 or more high-speed separations in parallel on a single wafer-scale device. The fluorescently labeled DNA samples are detected within the microchannels with a novel four-color rotary confocal fluorescence scanner. The capabilities of this system for genotyping are demonstrated through multiplex separations of short tandem repeat and hereditary haemochromatosis allele-specific amplicons. Furthermore, with newly developed folded channel designs that maintain high resolution, these CAE microplate systems are used to perform 96 high-quality DNA sequencing separations in parallel to approximately 500 bases per capillary in less than 30 min. These densely packed microfabricated device technologies will facilitate the even more rapid collection of vast amounts of genetic data in the future.

Turn geometry for minimizing band broadening in microfabricated capillary electrophoresis channels.

Turns in microfabricated capillary electrophoresis channels generally result in degraded separation quality. To circumvent this limitation, channels were constructed with different types of turns to determine the design that minimizes turn-induced band broadening. In particular, tapered turns were created by narrowing the separation channel width before the start of a turn and widening the channel after the turn is complete. The radius of curvature of the turn, the length over which the channel is tapered, and the degree of tapering were explored. The column efficiencies were determined by examining the resolution of the 271/281 base pair doublet in the separation of a phiX174 HaeIII DNA sizing ladder. Tapered turns with the smallest radius of curvature (250 microm), the shortest tapering length between the separation and turn widths (55 microm), and the largest tapering ratio (4:1 separation channel width to turn channel width) produced the highest resolution separations. These results are discussed by comparison to theoretical predictions of the effect of tapers and turns on analyte band dispersion in capillary electrophoresis.