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 ...

The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500-to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2-to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.Most living cells are sensitive to sudden heat exposure. A shift in temperature from a low to an intermediate temperature induces the stress response or heat-shock response (1-3), which is considered to be an evolutionarily conserved genetic system advantageous to living organisms. After a temperature shift from 23 to 37°C in cells of the yeast Saccharomyces cerevisiae, 80 proteins were transiently induced; 20 of these proteins are now classified as major heat-shock proteins (HSPs) (2). Some of these HSPs have been characterized, but the function of many of them is still unclear (4).Initial studies suggested that HSPs play an essential role in the acquisition of stress tolerance. On the other hand, a convincing involvement of HSPs in the development of stress resistance has not been established in yeast. Except for HSP104, none of the other HSP disruption mutants show any block in the acquisition of stress resistance in yeast (5). Furthermore, a yeast strain with a temperature-sensitive mutation in the heat-shock factor (hsfl-m3) that leads to a general block in heat-shock-induced protein synthesis was not affected in the acquisition of thermotolerance (6). Therefore, HSPs may not be important for stress tolerance acquisition but rather for a rapid recovery after heat shock (4).The main factors causing death after heat exposure are still unknown. Thus, the heat-shock response may not elucidate why cells die in response to heat exposure but rather how they repair the damage afterwards. To investigate why cells die in response to heat exposure, we completely avoided the induction of the heat-shock response by exposing cells immediately to lethal heat. In particular, we investigated the possible involvement of oxidative stres...

In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50°C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H 2 O 2 at 42 as at 30°C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H 2 O 2 . We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.

The theory presented here describes the motion of a large gas bubble rising through upward-flowing liquid in a tube. The basis of the theory is that the liquid motion round the bubble is inviscid, with an initial distribution of vorticity which depends on the velocity profile in the liquid above the bubble. Approximate solutions are given for both laminar and turbulent velocity profiles and have the form \begin{equation} U_s = U_c+(gD)^{\frac{1}{2}}\phi(U_c/(gD)^{\frac{1}{2}}), \end{equation}Us being the bubble velocity, Uc the liquid velocity at the tube axis, g the acceleration due to gravity, and D the tube diameter; ϕ indicates a functional relationship the form of which depends upon the shape of the velocity profile. With a turbulent velocity profile, a good approximation to (1) which is suitable for many practical purposes is \begin{equation} U_s = U_s + U_{s0}, \end{equation}Us0 being the bubble velocity in stagnant liquid. Published data for turbulent flow are known to agree with (2), so that in this case the theory supports a well-known empirical result. Our laminar flow experiments confirm the validity of (1) for low liquid velocities.

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