Search

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.

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.

Blends of poly(p-phenylenebenzo[1,2-d:5,4-d′]bisoxazole) (PBO) dispersed in poly(ether ether ketone) (PEEK) have been prepared by casting and coagulating methanesulfonic acid (MSA) solutions containing different fractions of each polymer. At PBO fractions greater than about 1%, the fluorescence spectra resemble that of ‘bulk’ PBO coagulates; but at lower PBO concentrations, the spectra show a sharper, blue-shifted peak, close to the 0-0 peak of dilute PBO/MSA. This emission most likely originates from PBO chains that have been isolated in a photophysical sense. This result, along with the solvent shift seen in a model compound, suggests that the deprotonation minimally perturbs the emission spectrum of PBO relative to the changes caused by aggregation.

The effect of compression on the fluorescence and Raman spectra of poly( p -phenylene benzobisoxazole) (PBO) fibre and a PBO/amorphous nylon blend has been investigated qualitatively at various excitation wavelengths. A substantial red-shift in the fluorescence maxima is observed upon increasing pressure both in PBO fibres as well as in the PBO/amorphous nylon blend. The shift is consistent with that observed for aromatic molecular crystals. This observation strongly suggests that certain features in the fluorescence emission are due to interchain interactions.

The photo-fluorescence of a solution-cast film containing a 30 70 blend of poly(p-phenylene benzobisoxazole) (PBO)/amorphous nylon shows a substantial decrease in fluorescence yield, and a blue-shifted spectrum upon annealing at around 220°C, corresponding to the transition from an entrapped single phase to a multiphase system. This indicates a substantial dependence of PBO fluorescence on its domain structure in this blend.

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 germinate 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 these geminate ion pairs.

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.

Time-resolved fluorescence-detected magnetic resonance (FDMR) studies of irradiated alkane solutions of trialkylamines and scintillators reveal the EPR spectra of the trialkylaminium radicals, formed by scavenging solvent radical cations. A qualitative kinetic analysis indicates that the growth of the triethylaminium radical (TEA/sup +/-) FDMR signal occurs on similar time scales in both n-hexane and cyclohexane, suggesting that, in cyclohexane, TEA/sup +/- is formed by scavenging the lower mobility ''trapped'' cyclohexane radical cations. Fluorescence results indicate that TEA quenches both scintillator fluorescence and total FDMR intensities to a greater extent than is expected from amine scavenging of solvent holes. TEA also exhibits an intense, relatively long-lived fluorescence which is apparently not produced by radical ion recombination or energy transfer. 6 figures