Stephanie H. I. Yeung

Integrated sample cleanup-capillary electrophoresis microchip for high-performance short tandem repeat genetic analysis.

An integrated PCR sample cleanup and preconcentration process is developed for forensic short tandem repeat (STR) analysis using a streptavidin-modified photopolymerized capture gel injector for microchip capillary electrophoresis (microCE). PCR samples generated with one biotinylated primer and one fluorescent primer provide the input to the streptavidin-based affinity capture-microCE device. Monoplex PCR samples processed by the device exhibited approximately 10- to 50-fold increased fluorescence intensities, and DNA profiles generated using 9-plex STR samples displayed approximately 14- to 19-fold higher signal intensities compared to those analyzed using traditional cross injection. Complete STR profiles were obtained with as few as 25 copies of DNA template using the capture-microCE device. Four DNA samples with various degrees of degradation were also tested. Samples analyzed using the capture-microCE device resulted in a significant increase of successful allele detection. The ability of our capture-microCE device and method to remove contaminating ions, to concentrate the sample injection plug, and to eliminate electrokinetic injection bias provides a powerful approach for integrating sample cleanup with DNA separation.

Fluorescence energy transfer-labeled primers for high-performance forensic DNA profiling.

A fluorescence energy transfer (ET) dye‐labeled STR typing system (ET 16‐plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96‐lane microfabricated capillary array electrophoresis (μCAE) system. The ET 16‐plex amplicons displayed 1.6–9‐fold higher fluorescence intensities compared to those produced using the single‐dye (SD)‐labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor‐quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16‐plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop‐in allele incidence. To confirm the practical advantages of ET‐labeled primers, six previously problematic casework samples were tested and only the ET 16‐plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the μCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher‐efficiency forensic laboratory enabled by better chemistry and microfluidics.

Rapid Determination of Monozygous Twinning with a Microfabricated Capillary Array Electrophoresis Genetic-Analysis Device

BACKGROUND Microfabricated genetic-analysis devices have great potential for delivering complex clinical diagnostic technology to the point of care. As a demonstration of the potential of these devices, we used a microfabricated capillary array electrophoresis (microCAE) instrument to rapidly characterize the familial and genotypic relationship of twins who had been assigned fraternal (dizygous) status at birth. METHODS We extracted the genomic DNA from buccal samples collected from the twin sons, the parents, another sibling, and an unrelated control individual. We then carried out multiplex PCR amplification of sequences at 16 short tandem repeat loci commonly used in forensic identity testing. We simultaneously separated the amplicons from all of the individuals on a microCAE device and fluorescently detected the amplicons with single-base resolution in <30 min. RESULTS The genotypic analysis confirmed the identical status of the twins and revealed, in conjunction with the medical data, that their twin status arose from the rarer dichorionic, diamniotic process. CONCLUSIONS The ability to rapidly analyze complex genetic samples with microCAE devices demonstrates that this approach can help meet the growing need for rapid genetics-based diagnostics.

Formation of Nanometer-Scale Dot Arrays from Diblock Copolymer Templates

We have developed four different fabrication processes based on self-organizing diblock copolymers that all produce densely-spaced, uniformly-sized nanometer-scale dot arrays over large wafer areas. We demonstrate the versatility of these techniques through examples of dot arrays formed of metallic, insulating, and polymeric materials. These fabrication processes vary in complexity, utility, and degree of optimization, and we discuss the relative merits of each. The ability to create uniform nanoscale features below lithographic resolution limits may enable key applications in fields such as magnetic recording and microelectronics.

Integrated Sample Cleanup and Microchip Capillary Array Electrophoresis for High-Performance Forensic STR Profiling

Microfluidics has the potential to significantly improve the speed, throughput, and cost performance of electrophoretic short tandem repeat (STR) analysis by translating the process into a miniaturized and integrated format. Current STR analysis bypasses the post-PCR sample cleanup step in order to save time and cost, resulting in poor injection efficiency, bias against larger loci, and delicate injection timing controls. Here we describe the operation of an integrated high-throughput sample cleanup and capillary array electrophoresis microsystem that employs a streptavidin capture gel chemistry coupled to a simple direct-injection geometry for simultaneously analyzing 12 STR samples in less than 30 min with >10-fold improved sensitivity.

High-performance forensic DNA profiling using fluorescence energy transfer primers and a 96-lane microfabricated capillary array electrophoresis device

We present a 96-channel microfabricated capillary array electrophoresis (muCAE) device for fluorescence energy- transfer (ET) dye labeled short tandem repeat (STR) genotyping. The 96-channel muCAE system produced high-speed (<30 min), highly parallel DNA separations with single-base resolution. To achieve higher sensitivity of STR typing, we adapted ET dye labeling method, and the resultant ET 16-plex amplicons which are the markers used in the commercial STR typing kit PowerPlex 16 displayed 1.6-8.7 fold increased fluorescence intensities and improved limit of detection compared to those produced using the single-dye labeled multiplex kit. In addition, the ET 16-plex generated complete STR profiles with only 28 PCR cycles; this capability should improve low copy number (LCN) typing by reducing the amplification time and background, thereby providing higher successful profiling rate. To confirm the practical advantages of ET-labeled primers, six previously problematic sexual casework samples were tested and only the ET 16-plex kit was able to capture additional DNA allele data. The successful development and demonstration of ET cassette labeled primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but also a significant step toward a higher-efficiency integrated forensic system.

Microfluidic Circuit for Integrated DNA Sequencing Product Purification and Analysis

A microfluidic circuit for DNA sequencing sample desalting, template removal, and preconcentration has been integrated with on-chip electrophoretic analysis. A 60-nL chamber containing a DNA affinity copolymer is used to capture and preconcentrate sequencing extension products in only 120 s. Thermal denaturation of the affinity hybrid is followed by high-speed microchip electrophoresis resulting in 560 bases with an accuracy ≥ 99% in only 30 min.