Molecular Diagnostics, Inc.

Geneohm Sciences, Inc.

Genohm Sciences

Donald M Crothers

Nanibhushan Dattagupta

Honghua Zhang

William J Knowles

Chunnian Shi

Peter M Rae

R Erik Holmlin

Thomas R Barnett

Vincent T Marchesi

Carol Koenigsberger

Erik R Holmlin

Disclosed herein are methods of destabilizing double-stranded nucleic acid hybridization using an enzyme comprising DNA N-glycosylase activity. Also disclosed herein is the detection of a double-stranded target DNA wherein the hybridization of duplex strands has been at least partially disrupted thereby permitting invasion of a probe strand. Also disclosed herein are methods of using an enzyme comprising DNA N-glycosylase activity to generate single-stranded circular nucleic acids.

Nucleic acid hybridization methods

The present disclosure provides methods and compositions for conducting an assay to detect a polynucleotide. In particular, ruthenium complexes having reduction potentials that do not coincide with the reduction potential of molecular oxygen are disclosed and amperometric techniques for their use are described. In preferred embodiments, the ruthenium complex is ruthenium (III) pentaamine pyridine and the polynucleotide that is detected is DNA. Further, techniques for enhancing detectable contrast between hybridized and unhybridized nucleic acids are disclosed. In particular, the use of elongated target strands as well as the use of uncharged probe strands are discussed.

Electrochemical method to measure DNA attachment to an electrode surface in the presence of molecular oxygen

The present disclosure provides methods and compositions for conducting an assay to detect nucleic acid hybridization in the presence of oxygen. In particular, ruthenium complexes having a reduction potential that does not coincide with the reduction potential of molecular oxygen are disclosed and amperometric techniques for their use are described. In preferred embodiments, the ruthenium complex is ruthenium (III) pentaamine pyridine and the nucleic acid hybridization event that is detected is DNA hybridization. Further, techniques for enhancing detectable contrast between hybridized and unhybridized nucleic acids are disclosed. In particular, the use of elongated target strands as well as the use of uncharged probe strands are discussed.

The present disclosure relates to methods for generating single-stranded DNA molecules of defined sequence and length. Specifically, a region of template containing target sequence is amplified by PCR or RCA, exogenous sequence is introduced by primers or probes used in amplification, double-stranded amplification products are converted to single-stranded amplification products, and single-stranded amplification products are trimmed to produce short single-stranded DNA molecules of defined sequence and length.

Amplification of DNA to produce single-stranded product of defined sequence and length

A labeled nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to the nucleic acid-binding ligand. The label can be a specifically bindable ligand such as a hapten or biotin, an enzyme such as a .beta.-galactosidase or horse radish peroxidase, a fluorescent radical, a phycobiliprotein, a luminescent radical, or a radioisotope. The probe can be used in assays of nucleic acids, taking advantage of the ability of the nucleic acid component to hydridize.

Fast photochemical method of labelling nucleic acids for detection

A nucleic acid detection probe comprising a hybridizable single stranded portion of nucleic acid connected with a non-hybridizable, single or double stranded nucleic acid portion, the non-hybridizable portion preferably including a recognition site for binding by a particular protein. Such recognition site can be a region of singly or doubly stranded nucleic acid specific for a particular nucleic acid binding protein such as lac repressor protein or can be a modified nucleic acid region such as a unique antigenic determinant introduced by interaction of the region with a modifier compound such as an intercalating agent or a platinum-containing ligand. The probe-binding protein can be labeled for ease of detection and in the case of an antigenic determinant binding site can be labeled antibody.

Nucleic acid probe detectable by specific nucleic acid binding protein

A protein is covalently coupled to a 3'terminal end of a nucleic acid which carries several labels. In an assay the protein will specifically recognize some component of a test system; in an immunoassay the protein can be Protein A which will recognize the FC portion of IgG which is bound to an unknown antigen if present in the test sample.

Nucleic acid-protein conjugate used in immunoassay

A labeled nucleic acid probe comprising (a) a nucleic acid component, (b) a nucleic acid-binding ligand photochemically linked to the nucleic acid component, and (c) a label chemically linked to the nucleic acid-binding ligand. The label can be a specifically bindable ligand such as a hapten or biotin, an enzyme such as a .beta.-galactosidase or horse radish peroxidase, a fluorescent radical, a phycobiliprotein, a luminescent radical, or a radioisotope. The probe can be used in assays of nucleic acids, taking advantage of the ability of the nucleic acid component to hybridize.

A process for production of a single strand of a nucleic acid comprising covalently linking to a solid substrate a polynucleotide complementary to the desired strand, hybridizing said polynucleotide with an oligonucleotide, extending the oligonucleotide in direction away from said substrate, denaturing the hybridized polynucleotide and extended oligonucleotide, thereby to free the extended oligonucleotide from the solid substrate, and separating the extended oligonucleotide. The product can be used for making analytical probes.

Large scale production of DNA probes

A detection probe comprising a hybridizable single stranded portion of nucleic acid connected with a non-hybridizable, single or double stranded nucleic acid portion, the non-hybridizable portion preferably including a recognition site for a particular protein.

Use of non-hybridizable nucleic acids for the detection of nucleic acid

A solid support capable of binding a nucleic acid thereto upon suitable irradiation, comprising (a) a solid substrate, (b) a photochemically reactive intercalator compound or other nucleic acid-binding ligands, and (c) divalent radical chemically linking the substrate and the ligand (b). Specifically, a hydroxy group-containing solid substrate such as nitrocellulose paper is linked via a bifunctional reagent such as cyanogen bromide or 1,4-butanedioldiglycidyl ether to an amino-substituted angelicin or psoralen or ethidium bromide which in turn is photochemically linked to a nucleic acid. The resulting immobilized nucleic acid probe is capable of hybridizing with complementary nucleic acid fragments and is thereby useful in diagnostic assays.

Coupling of nucleic acids to solid support by photochemical methods

A solid support capable of binding a nucleic acid thereto upon suitable irradiation, comprising (a) a solid substrate, (b) a member selected from the group consisting of a furocoumarin, a phenanthridium halide, and photochemically reactive derivatives thereof, and (c) a divalent radical chemically linking the substrate and the member (b). Specifically, a hydroxy group-containing solid substrate such as nitrocellulose paper is linked via a bifunctional reagent such as cyanogen bromide or 1,4-butanediol diglycidyl ether to an amino-substituted angelicin or psoralen or phenanthridinium bromide which in turn is photochemically linked to a nucleic acid. This is capable of hybridizing with other nucleic acid fragments and is thereby useful in diagnostic assays.

Growing community of inventors

Northford, CT, United States of America

Donald M Crothers