The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.DNA sequencing has markedly changed the nature of biomedical research and medicine. Reductions in the cost, complexity and time required to sequence large amounts of DNA, including improvements in the ability to sequence bacterial and eukaryotic genomes, will have significant scientific, economic and cultural impact. Largescale sequencing projects, including whole-genome sequencing, have usually required the cloning of DNA fragments into bacterial vectors, amplification and purification of individual templates, followed by Sanger sequencing 1 using fluorescent chain-terminating nucleotide analogues 2 and either slab gel or capillary electrophoresis. Current estimates put the cost of sequencing a human genome between $10 million and $25 million 3 . Alternative sequencing methods have been described 4-8 ; however, no technology has displaced the use of bacterial vectors and Sanger sequencing as the main generators of sequence information.Here we describe an integrated system whose throughput routinely enables applications requiring millions of bases of sequence information, including whole-genome sequencing. Our focus has been on the co-development of an emulsion-based method 9-11 to isolate and amplify DNA fragments in vitro, and of a fabricated substrate and instrument that performs pyrophosphate-based sequencing (pyrosequencing 5,12 ) in picolitre-sized wells.In a typical run we generate over 25 million bases with a Phred quality score of 20 or better (predicted to have an accuracy of 99% or higher). Although this Phred 20 quality throughput is significantly higher than that of Sanger sequencing by capillary electrophoresis, it is currently at the cost of substantially shorter reads and lower average individual read accuracy. Sanger-based capillary electrophoresis sequencing systems produce up to 700 bases of sequence information from each of 96 DNA templates at an average read accuracy of 99.4% in 1 h, or 67,000 bases per hour, with substantially all of the bases having Phred 20 or better quality 23 . We further characterize the performance ...

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Gold has been the metal of choice for research on molecular tunneling junctions, but it is incompatible with CMOS fabrication because it forms deep level traps in silicon. Palladium electrodes do not contaminate silicon, and also give higher tunnel current signals in the molecular tunnel junctions we have studied. The result is cleaner signals in a recognition-tunneling junction that recognizes the four natural DNA bases as well as 5-methyl cytosine, with no spurious background signals. More than 75% of all the recorded signal peaks call the base correctly.

Time-resolved fluorescence-detected EPR studies of electron-irradiated solutions of 2,5-diphenyloxazole (PPO) in alkane solvents have shown that there are two distinct geminate radical ion pairs, distinguished by their lifetimes and EPR spectra. While the central EPR peak belongs to the aromatic radical ions, the broader components are assigned to the parent radical cation of the alkane solvent. Analysis of the resolved hyperfine structure indicates the presence of a trapped solvent hole in cyclohexane, which does not undergo unusually rapid charge exchange. The kinetic analyses of the EPR spectra show that scavenging events preserve the relative orientation of the unpaired electron spins in

The optically detected magnetic resonance (ODMR) spectra of the covalent DNA adduct of 9,10-epoxy-9,10,11,12-tetrahydrobenzo[e]pyrene (BePE) reveal that the excited triplet state chromophore is perturbed by the nucleic acid and that this perturbation is diminished successively by denaturation and enzymatic hydrolysis of the modified DNA, indicating that the adduct resides in an environment with some quasi-intercalative character. In contrast the covalent adduct of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene shows little ODMR evidence of interaction with the nucleic acid, which, in view of ODMR's sensitivity demonstrated for the BePE adduct, suggests that the chromophore is situated in an environment resembling the bulk solvent. These results demonstrate that ODMR is more sensitive than conventional phosphorescence techniques to interactions between these pyrene-like chromophores and DNA.

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