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.

Real-time forensic DNA analysis at a crime scene using a portable microchip analyzer.

An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2h and 30min with

A forensic laboratory tests the Berkeley microfabricated capillary array electrophoresis device.

Miniaturization of capillary electrophoresis onto a microchip for forensic short tandem repeat analysis is the initial step in the process of producing a fully integrated and automated analysis system. A prototype of the Berkeley microfabricated capillary array electrophoresis device was installed at the Virginia Department of Forensic Science for testing. Instrument performance was verified by PowerPlex 16 System profiling of single source, sensitivity series, mixture, and casework samples. Mock sexual assault samples were successfully analyzed using the PowerPlex Y System. Resolution was assessed using the TH01, CSF1PO, TPOX, and Amelogenin loci and demonstrated to be comparable with commercial systems along with the instrument precision. Successful replacement of the Hjerten capillary coating method with a dynamic coating polymer was performed. The accurate and rapid typing of forensic samples demonstrates the successful technology transfer of this device into a practitioner laboratory and its potential for advancing high-throughput forensic typing.

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.

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 (muCAE) 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 muCAE 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 and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice.

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.

Sparsely cross-linked "nanogel" matrixes as fluid, mechanically stabilized polymer networks for high-throughput microchannel DNA sequencing.

We have developed sparsely cross-linked "nanogels", subcolloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel replaceable DNA sequencing matrixes for capillary and microchip electrophoresis. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and a low percentage (approximately 10(-4) mol %) of N,N-methylene bisacrylamide (Bis). Nanogels and nanogel networks were characterized by multiangle laser light scattering and rheometry, respectively, and tested for DNA sequencing in both capillaries and chips with four-color LIF detection. Typical nanogels have an average radius of approximately 230 nm, with approximately 75% of chains incorporating a Bis cross-linker. The properties and performance of nanogel matrixes are compared here to those of a linear polyacrylamide (LPA) network, matched for both polymer weight-average molar mass (M(w)) and the extent of interchain entanglements (c/c). At sequencing concentrations, the two matrixes have similar flow characteristics, important for capillary and microchip loading. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, substantially longer average read lengths are obtained under standard conditions with the nanogel matrix at a 98.5% accuracy of base-calling (for CE: 680 bases, an 18.7% improvement over LPA, with the best reads as long as 726 bases, compared to 568 bases for the LPA matrix). We further investigated the use of the nanogel matrixes in a high-throughput microfabricated DNA sequencing device consists of 96 separation channels densely fabricated on a 6-in. glass wafer. Again, preliminary DNA sequencing results show that the nanogel matrixes are capable of delivering significantly longer average read length, compared to an LPA matrix of comparable properties. Moreover, nanogel matrixes require 30% less polymer per unit volume than LPA. The addition of a small amount of low molar mass LPA or ultrahigh molar mass LPA to the optimized nanogel sequencing matrix further improves read length as well as the reproducibility of read length (RSD < 1.6%). This is the first report of a replaceable DNA sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrixes appropriately matched for molar mass and the extent of interchain entanglements. These results could have significant implications for the improvement of microchip-based DNA sequencing technology.

Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing.

A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3' of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50 degrees C, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67 degrees C and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality q > or = 20 (accuracy > or = 99%). This fully integrated nanoliter process decreases the purification time approximately 10-fold and the process volume approximately 100-fold while providing state-of-the-art sequencing results.