Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is approximately 20 A away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R(P) oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd(2+) rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd(2+) concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd(2+) is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tight-binding metal ion (< 10 microM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd(2+) binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd(2+) is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd(2+)-rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.

Highly uniform and conformal coatings can be made by the alternating exposures of a surface to vapors of two reactants, in a process commonly called atomic layer deposition (ALD). The application of ALD has, however, been limited because of slow deposition rates, with a theoretical maximum of one monolayer per cycle. We show that alternating exposure of a surface to vapors of trimethylaluminum and tris(tert-butoxy)silanol deposits highly conformal layers of amorphous silicon dioxide and aluminum oxide nanolaminates at rates of 12 nanometers (more than 32 monolayers) per cycle. This process allows for the uniform lining or filling of long, narrow holes. We propose that these ALD layers grow by a previously unknown catalytic mechanism that also operates during the rapid ALD of many other metal silicates. This process should allow improved production of many devices, such as trench insulation between transistors in microelectronics, planar waveguides, microelectromechanical structures, multilayer optical filters, and protective layers against diffusion, oxidation, or corrosion.

This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.

Zinc-dependent histone deacetylase 8 (HDAC8) catalyzes the removal of acetyl moieties from histone tails, and is critically involved in regulating chromatin structure and gene expression. The detailed knowledge of its catalytic process is of high importance since it has been established as a most promising target for the development of new anti-tumor drugs. By employing BornOppenheimer ab initio QM/MM molecular dynamics simulations and umbrella sampling, a stateof-the-art approach to simulate enzyme reactions, we have provided further evidences against the originally proposed general acid-base catalytic pair mechanism for Zinc-dependent histone deacetylases. Instead, our results indicated that HDAC8 employs a proton-shuttle catalytic mechanism, in which a neutral His143 first serves as the general base to accept a proton from the zinc-bound water molecule in the initial rate-determining nucleophilic attack step, and then shuttles it to the amide nitrogen atom to facilitate the cleavage of the amide bond. During the deacetylation process, the Zn 2+ ion changes its coordination mode and plays multiple catalytic roles. For the K + ion, which is located about 7 Å from the catalytic Zn 2+ ion and conserved in class I and II HDACs, our simulations indicated that its removal would lead to the different transition state structure and a higher free energy reaction barrier for the rate-determining step. It is found that the existence of this conserved K + ion would enhance the substrate binding, increase the basicity of His143, strengthen the catalytic role of zinc ion and improve the transition state stabilization by the enzyme environment.