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Journal ArticleDOI

A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes.

10 Jun 1988-Nucleic Acids Research (Oxford University Press)-Vol. 16, Iss: 11, pp 4937-4956
TL;DR: The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.
Abstract: N4-[N-(6-trifluoroacetylamidocaproyl)-2-aminoethyl]-5'-O-dimethoxy trityl -5-methyl-2'-deoxycytidine-3'-N,N-diisopropyl-methylphosphoramidite++ + has been synthesized. This N4-alkylamino deoxycytidine derivative has been incorporated into oligonucleotide probes during chemical DNA synthesis. Subsequent to deprotection and purification, fluorescent (fluorescein, Texas Red and rhodamine), chemiluminescent (isoluminol), and enzyme (horseradish peroxidase, alkaline phosphatase) labels have been specifically incorporated. Detection limits of the labels and labeled probes were assessed. Also, the detection limits and nonspecific binding of the labeled probes in sandwich hybridization assays were determined. The enzyme modified oligonucleotides were found to be significantly better labeling materials than the fluorescent or chemiluminescent derivatives, providing sensitivities comparable to 32P-labeled probes.
Citations
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Journal ArticleDOI
22 Aug 1997-Science
TL;DR: A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported, which can detect about 10 femtomoles of an oligonucleotide.
Abstract: A highly selective, colorimetric polynucleotide detection method based on mercaptoalkyloligonucleotide-modified gold nanoparticle probes is reported. Introduction of a single-stranded target oligonucleotide (30 bases) into a solution containing the appropriate probes resulted in the formation of a polymeric network of nanoparticles with a concomitant red-to-pinkish/purple color change. Hybridization was facilitated by freezing and thawing of the solutions, and the denaturation of these hybrid materials showed transition temperatures over a narrow range that allowed differentiation of a variety of imperfect targets. Transfer of the hybridization mixture to a reverse-phase silica plate resulted in a blue color upon drying that could be detected visually. The unoptimized system can detect about 10 femtomoles of an oligonucleotide.

4,334 citations

Patent
07 Jun 1990
TL;DR: In this paper, a method and apparatus for preparation of a substrate containing a plurality of sequences is described, where a set of photoremovable groups are attached to a surface of the substrate and selected regions of the surface are exposed to light so as to activate the selected areas.
Abstract: A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

1,171 citations

Patent
25 Jun 1996
TL;DR: In this article, a method and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers, was presented, which can also be used for classification of biological samples, and to characterize their sources.
Abstract: The present invention provides method and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

869 citations

Patent
20 Nov 1992
TL;DR: In this article, a method and device for forming large arrays of polymers on a substrate was proposed, where the substrate is contacted by a channel block having channels and selected reagents are delivered through the channels.
Abstract: A method and device for forming large arrays of polymers on a substrate ( 401 ). According to a preferred aspect of the invention, the substrate is contacted by a channel block ( 407 ) having channels ( 409 ) therein. Selected reagents are delivered through the channels, the substrate is rotated by a rotating stage ( 403 ), and the process is repeated to form arrays of polymers on the substrate. The method may be combined with light-directed methodolgies.

848 citations

Book ChapterDOI
TL;DR: The chapter discusses FRET specifying the physical parameters that are available from a variety of fluorescence measurements, and some basic considerations and experimental procedures are described that are necessary to make reliable FRET measurements.
Abstract: Publisher Summary Fluorescence resonance energy transfer (FRET) is a spectroscopic process by which energy is passed nonradiatively among molecules over long distances. The “donor” molecule, which must be a fluorophore, absorbs a photon and transfer this energy nonradiatively to the “acceptor” molecule. Energy can be transferred over distances on the order of common macromolecular dimensions. This chapter discusses the applications of FRET to nucleic acid molecules conjugated to dye molecules; but in general, the donor-acceptor pairs can be free in solution, one or both bound to a macromolecule, or be an inherent part of the structure. The chapter discusses FRET specifying the physical parameters that are available from a variety of fluorescence measurements. In the chapter, some basic considerations and experimental procedures are described that are necessary to make reliable FRET measurements. It also discusses some difficulties that can arise in the experimental proceedings and data analysis and the sources of experimental error are pointed out.

766 citations

References
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Journal ArticleDOI
TL;DR: This work has shown that effective methods of peptide synthesis are crucial to progress in this area, because only by synthesis can adequate amounts of important peptides be made available for chemical, biologi...
Abstract: The last two decades have been an era of rapid progress in peptide research. This era was begun by the work of Sanger on the amino acid sequence determination of insulin and by du Vigneaud on the structure determination and synthesis of oxytocin. This period has seen impressive progress in the structure elucidation and synthesis of many peptides of natural origin and of great biological significance, as well as in methods for sequence determination and chemical synthesis of peptides [1–4]. Perfection of techniques and instruments for automatic determination of the amino acid sequence of peptides and proteins has made possible a greatly broadened understanding of genetics and evolution as well as the more chemical areas of mechanism of action of enzymes and hormones, and physical chemistry of peptides and proteins. Effective methods of peptide synthesis are crucial to progress in this area, because only by synthesis can adequate amounts of important peptides be made available for chemical, biologi...

3,519 citations

01 Jan 1985
TL;DR: This book contains papers on Using Nucleic Acid Filter Hybridization to Characterize and Detect Enteroviral RNAs, Rapid Identification of Lesihmania Species using Specific Hybridization of Kinetoplast DNA Sequences, and Selection of DNA Probes for use in the Diagnosis of Infectious Disease.
Abstract: This book contains papers divided among five sections. Some of the paper titles are: Aspects of Using Nucleic Acid Filter Hybridization to Characterize and Detect Enteroviral RNAs; Rapid Identification of Lesihmania Species using Specific Hybridization of Kinetoplast DNA Sequences; Selection of DNA Probes for use in the Diagnosis of Infectious Disease; and Summary of DNA Probes.

102 citations

Journal ArticleDOI
TL;DR: This volume is an important attempt to bridge the conceptual gap between Dr Seegers' own work and that of others, and if read in conjunction with the recent reviews of Macfarlane and of Biggs it leaves no one with the excuse that he cannot at least begin to understand Dr SeEGers' work.
Abstract: activity in concentrated salt solution. Chapters V and VI deal with the clotting defects of oral anticoagulants and of congenital bleeding disorders respectively and a long final chapter discusses the nature of serum. This volume is an important attempt to bridge the conceptual gap between Dr Seegers' own work and that of others, especially if read in conjunction with the recent reviews of Macfarlane and of Biggs (Thromb. Diath. haem., 15, 591 and 603, 1966) it leaves no one with the excuse that he cannot at least begin to understand Dr Seegers' work.

26 citations