DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally employed long (400–800 bp) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intra-species genetic variation. We report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterise four million SNPs and four hundred thousand structural variants, many of which are previously unknown. Our approach is effective for accurate, rapid and economical whole genome re-sequencing and many other biomedical applications.
Semiconductor nanocrystals or quantum dots (QDs) are highly photoluminescent materials with unique optical attributes that are being exploited in an ever-increasing array of applications. However, the complex surface chemistry of these finite-sized fluorophores gives rise to a number of photophysical phenomena that can complicate their use in imaging applications. Fluorescence intermittency (FI), photoluminescence enhancement (PLE) and spectral bluing are properties of QD emission that would appear, at first sight, detrimental to quantitative measurement. Fortunately, developments in rational QD synthesis and surface modification are promising to minimize the effects of these fluorescence instabilities, while applications that exploit them are now coming to the fore. We review recent experimental and theoretical studies of FI, PLE and bluing, highlighting the benefits, as well as complications, they bring to key applications.
We have used confocal fluorescence microscopy to study the diffusion of single molecules in solution, over a wide dynamic mass range. The results have been analyzed using both Poisson and autocorrelation methods and have been shown to be consistent with diffusion theory over extended time scales. We observe deviations from statistical behavior on short time scales due to a weak optically induced trapping effect. The power dependency of this effect has been measured and shown to be in good agreement with optical trapping theory. We find that for small molecules the optical biasing effect is only significant for molecules excitated at resonance.
We have re-examined critical experiments on collision induced rotational transfer (RT) and conclude that the probability of RT is controlled by the factors that control the probability of angular momentum (AM) change. The probability of energy change seems less important in this respect. In the light of this we suggest a model for RT in which the probability of AM change is calculated directly and present a formalism for this purpose. We demonstrate that such a calculation leads to an exponential-like fall of RT probabilities with transferred AM, a consequence of the radial dependence of the repulsive part of the intermolecular potential. Thus in this AM model, the exponential gap law has a simple physical origin. The AM model we describe may be used as the basis of an inversion routine through which it is possible to convert RT data into a probability density of the repulsive anisotropy. Through this model therefore it is possible to relate experimental RT data directly to the forces that are responsible for rotational transfer. The hard ellipse model is used in this work to relate calculated anisotropies to a form that includes an isotropic component. The result is a representation of the intermolecular potential through which new insights into the RT process are gained.
We have formulated a law for state-to-state rotational transfer (RT) in diatomic molecules based on the angular momentum (AM) theory proposed by McCaffery et al. [J. Chem. Phys. 98, 4586 (1993)]. In this, the probability of angular momentum change in the rotor is calculated by assuming the dominant process to be the conversion of linear to angular momentum at the repulsive wall of the intermolecular potential. The result is a very simple expression containing three variable parameters, each of which has physical significance in the context of the model. Fits to known RT data are very good and suggest strongly that linear to angular momentum change is indeed the controlling process in RT. The parameters of the fit are sufficiently available to give the model predictive power. Using this formulation, RT probabilities may be calculated for an unknown system with little more than the atomic masses, bond length, and velocity distribution. We feel that this represents an important step in the development of a simple physical picture of the RT process.
The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and by small inhibitory proteins that associate with the R1 catalytic subunit. In addition, the subcellular localization of the R2 subunit is regulated through the cell cycle and in response to DNA damage. We show that the fission yeast small RNR inhibitor Spd1 is intrinsically disordered and regulates R2 nuclear import, as predicted by its relationship to Saccharomyces cerevisiae Dif1. We demonstrate that Spd1 can interact with both R1 and R2, and show that the major restraint of RNR in vivo by Spd1 is unrelated to R2 subcellular localization. Finally, we identify a new behavior for RNR complexes that potentially provides yet another mechanism to regulate dNTP synthesis via modulation of RNR complex architecture.
We have employed single molecule fluorescence spectroscopy, using a total internal reflection geometry and wide-angle detection, to study the attachment of singly fluorescently labeled DNA to a silica surface by either a streptavidin-biotin or a covalent linkage. In both cases the DNA is highly monodispersed with no evidence for aggregation. The covalent coupling gave higher signal-to-noise than the streptavidin-biotin linkage and was therefore studied in more detail. Two components in the photobleaching times, corresponding to different states of the tetramethyl rhodamine probe, were observed: a short and long component with populations in the ratio 6.7:1. Only rarely was interconversion between these two states detected during the 30-s observation time of the experiment. Hybridization experiments using a complementary strand of DNA labeled with a different fluorophore gave a low level of colocalized fluorescence, indicating a significant fraction of the surface attached DNA was not available for hybridization. These results are consistent with the surface attached DNA spending significant time collapsed on the surface.
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