A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3′-OH unprotected cleavable fluorescent 2′-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor® 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA template

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm2 from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studie

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies

A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis†

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3′-OH unprotected cleavable fluorescent 2′-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor® 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal–no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates

Long-Term Memory Search across the Visual Brain

Signal transmission from the human retina to visual cortex and connectivity of visual brain areas are relatively well understood. How specific visual perceptions transform into corresponding long-term memories remains unknown. Here, I will review recent Blood Oxygenation Level-Dependent functional Magnetic Resonance Imaging (BOLD fMRI) in humans together with molecular biology studies (animal models) aiming to understand how the retinal image gets transformed into so-called visual (retinotropic) maps. The broken object paradigm has been chosen in order to illustrate the complexity of multisensory perception of simple objects subject to visual —rather than semantic— type of memory encoding. The author explores how amygdala projections to the visual cortex affect the memory formation and proposes the choice of experimental techniques needed to explain our massive visual memory capacity. Maintenance of the visual long-term memories is suggested to require recycling of GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and β2-adrenoreceptors at the postsynaptic membrane, which critically depends on the catalytic activity of the N-ethylmaleimide-sensitive factor (NSF) and protein kinase PKMζ

Interaction of pyridine with photoelectrons emitted at the silver/aqueous solution interface

Pyridine added to a solution containing scavengers of photoelectrons emitted from silver (N2O, CO2) causes a strong decrease in the measured photocurrent or even its complete suppression for concentrations of pyridine exceeding 0.1 M. Such behaviour, which remains in apparent contradiction to the effect of deposited pyridine on photoemission from silver in vacuum, is due to the fact that pyridine itself acts as an acceptor of the emitted photoelectrons. As the anion radical formed undergoes reoxidation (back electron transfer) at the silver surface, this process leads to a decrease in the net photocurrent which is dependent on the concentration ratio of the two competing acceptors (i.e. pyridine and N2O). The observation that pyridine (adsorbed on silver and in solution) sustains a reversible electron transfer, along with the previous observation that photoemission from a roughened Ag electrode into an aqueous solution extends to at least 650 nm, provides a possible interpretation of the surface-enhanced Raman spectroscopy effect for pyridine, which involves both electromagnetic and charge-transfer mechanisms

Conversion of amine- to carboxyl groups on solid surfaces

This invention provides a new method of obtaining a high density, reproducible and uniform coverage of a solid surface, compounds suitable for such a method and methods of preparing such compounds. This invention further relates to methods of the chemical modification (carboxylation) of solid surfaces and their subsequent use for the attachment of amine-containing molecules including DNA, proteins and other polymer

Improved method of nucleotide detection

The invention relates to an additive which can be added to buffers used in nucleotide detection processes and improved methods of nucleic acid sequencing using this additive. In particular the invention relates to use of the additive to improve the efficiency of fluorescence-based multiple cycle nucleic acid sequencing reaction

Method of Nucleotide Detection

The invention relates to an additive which can be added to buffers used in nucleotide detection processes and improved methods of nucleic acid sequencing using this additive. In particular the invention relates to use of the additive to improve the efficiency of fluorescence-based multiple cycle nucleic acid sequencing reaction

Electrochemical Reduction of Cyanides at Metallic Cathodes: A Comparison with Biological HCN Reduction

The electrochemical reduction of cyanides has been studied at a number of cathodes both in near-neutral and in alkaline solutions. Nickel appears as the most effective cathode material, promoting cyanide reduction with current efficiencies close to 70%, even in moderately alkaline solutions. In all cases, the eletroreduction of cyanides leads to a mixture of (methylamine) and products (methane and ammonia). The use of Nafion films loaded with Ni microparticles enabled us to markedly increase the effective current densities for cyanide reduction. The electrochemical reduction of is shown to present interesting similarities and differences with the biological cyanide reduction process. </p