The human genome will be sequenced using capillary array electrophoresis technology. Although currently achieving only 550 base reads per run, capillary arrays have increased the efficiency and lowered the cost of sequencing by eliminating gel plate preparation, reducing sample volumes, and offering automation and speed. However, much higher throughput and greater cost reductions are needed. The next major advancement in sequencing technology is expected from the development of arrays of microfabricated channels in a plate or "chip" format. For de novo sequencing, the practical utility of the microdevice approach has been limited by device length to a read of 500-600 bases per run. We demonstrate a significant milestone for a microfabricated device by obtaining an average read length of 800 bases in 80 min (98% accuracy) for either M13 standards or DNA sequencing samples from the Whitehead Institute Center for Genomic Research (WICGR) production line. This result is achieved in 40-cm-long channels using a new class of large-scale microfabricated devices. Both microfabrication of extended structures and achievement of long reads are essential steps toward a 384-lane very-large-scale microfluidic (VLSMF) system for ultrahigh-throughput DNA sequencing analysis, currently under construction in our laboratory.

A 768-lane DNA sequencing system based on microfluidic plates has been designed as a near-term successor to 96-lane capillary arrays. Electrophoretic separations are implemented for the first time in large-format (25 cm x 50 cm) microdevices, with the objective of proving realistic read length, parallelism, and the scaled sample requirements for long-read de novo sequencing. Two 384-lane plates are alternatively cycled between electrophoresis and regeneration via a robotic pipettor. A total of greater than 172000 bases, 99% accuracy (corresponding to quality score 20) is achieved for each iteration of a 384 lane plate. At current operating conditions, this implies a system throughput exceeding 4 megabases of raw sequence (Phred 20) per day on the new platform. Standard operation is at "1/32x" Sanger chemistry, equal to typical genome center operation on mature capillary array machines, and a 16-fold improvement in scaling relative to previous microfabricated devices. Experiments provide evidence that sample concentration can be further reduced to 1/256x Sanger chemistry in the microdevice. Life-testing indicates a usable life of >150 hours (more than 50 runs) for the 384 lane plates. The combined advances, particularly those in read length and sample requirement, directly address the cost model requirements for adaptation of the new technology as the next step beyond capillary array instruments.

We report preliminary testing of "GeneTrack", an instrument designed for the specific application of multiplexed short tandem repeat (STR) DNA analysis. The system supports a glass microdevice with 16 lanes of 20 cm effective length and double-T cross injectors. A high-speed galvanometer-scanned four-color detector was specially designed to accommodate the high elution rates on the microdevice. All aspects of the system were carefully matched to practical crime lab requirements for rapid reproducible analysis of crime-scene DNA evidence in conjunction with the United States DNA database (CODIS). Statistically significant studies demonstrate that an absolute, three-sigma, peak accuracy of 0.4-0.9 base pair (bp) can be achieved for the CODIS 13-locus multiplex, utilizing a single channel per sample. Only 0.5 microL of PCR product is needed per lane, a significant reduction in the consumption of costly chemicals in comparison to commercial capillary machines. The instrument is also designed to address problems in temperature-dependent decalibration and environmental sensitivity, which are weaknesses of the commercial capillary machines for the forensics application.

As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in approximately 100 s. This is 10 and 50 times faster than capillary and slab gel electro-phoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only +/-5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.

We present a new method for simplified low-quantity DNA loading onto microelectrophoresis devices. The method is based on combined solid-phase extraction, purification, and transport of DNA reversibly bound on paramagnetic microspheres. DNA is adsorbed onto the microspheres, captured with a magnetized permalloy wire, and then directly injected as a highly focused sample plug into the separation channel. This method circumvents both the minimum volume requirement of pipettors (since only solid beads are transported) and the timing complications of double-T microfluidic injection. Injections from Sanger samples of <100 pg total suspended weight match the signal strength of our previous conventional injections at >10-times the starting DNA sample. Sequencing traces show a resolution that matches or exceeds double-T injections. A kinetic model reproduces the time-dependence of the injection signals and proves that total nonidealities in the method produce injection-broadened plugs of approximately 1-s duration. The method should be broadly extendable to DNA and protein separations in both microdevice and capillary electrophoresis.