The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.DNA sequencing and, more recently, massively parallel DNA sequencing 1-4 has had a profound impact on research and medicine. The reductions in cost and time for generating DNA sequence have resulted in a range of new sequencing applications in cancer 5,6 , human genetics 7 , infectious diseases 8 and the study of personal genomes 9-11 , as well as in fields as diverse as ecology 12,13 and the study of ancient DNA 14,15 . Although de novo sequencing costs have dropped substantially, there is a desire to continue to drop the cost of sequencing at an exponential rate consistent with the semiconductor industry's Moore's Law 16 as well as to provide lower cost, faster and more portable devices. This has been operationalized by the desire to reach the $1,000 genome 17 .To date, DNA sequencing has been limited by its requirement for imaging technology, electromagnetic intermediates (either X-rays 18 , or light 19 ) and specialized nucleotides or other reagents 20 . To overcome these limitations and further democratize the practice of sequencing, a paradigm shift based on non-optical sequencing on newly developed integrated circuits was pursued. Owing to its scalability and its low power requirement, CMOS processes are dominant in modern integrated circuit manufacturing 21 . The ubiquitous nature of computers, digital cameras and mobile phones has been made possible by the low-cost production of integrated circuits in CMOS.Leveraging advances in the imaging field-which has produced large, fast arrays for photonic imaging 22 -we sought a suitable electronic sensor for the construction of an integrated circuit to detect the hydrogen ions that would be released by DNA polymerase 23 during sequencing by synthesis, as opposed to a sensor designed for the detection of photons. Although a variety ...

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Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes on the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.

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The use of arrays of immobilized DNA “probes” for high-throughput analysis of genomic samples is expanding rapidly. The detection sensitivity of these arrays depends on the quantity and density of immobilized probe molecules as well as on the thermodynamics and kinetics of nucleic acid hybridization. We have prepared and investigated substrates with a porous, “three-dimensional” surface layer as a means of increasing the surface area available for the synthesis or immobilization of oligonucleotide probes, thereby increasing the number of available probes and the amount of detectable bound target per unit area. Surfaces with pores 5 nm and larger were created by spin-coating colloidal suspensions of silica particles, followed by thermal curing. DNA arrays were synthesized on the resulting surfaces by photolithographic patterning, and the performance on the high-capacity substrates was compared to that on standard flat glass surfaces. The colloidal silica films created via this route show equivalent performance to flat glass substrates in terms of the efficiency of chemical synthesis and resolution of photolithographic patterning. DNA targets are able to penetrate the porous layers, and under saturating conditions, the quantity of bound target is proportional to the layer thickness. The result is an enhanced hybridization signal that is 20 times higher than flat glass for a colloidal particle layer that is 0.5 μm thick. The thermodynamic stability of probe/target duplexes in the matrix is the same as that for their counterparts on flat surfaces, although the colloidal silica films reach saturation more slowly than flat surfaces.

High-density DNA probe arrays provide a massively parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research and diagnostics. Light-directed combinatorial oligonucleotide synthesis has enabled the large-scale production of GeneChip probe arrays which contain several hundred of thousand oligonucleotide sequences on glass "chips" about one cm2 in size. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms ("genotyping"), and in a wide range of gene expression studies. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques adapted from the microelectronics industry. The present-generation methodology employs MeNPOC photo-activatable nucleoside monomers with proximity photolithography, and is currently capable of printing individual 10 microns 2 probe features at a density of 10(6) probes/cm2.