Beckman Instruments, Inc.

Beckman Coulter, Inc.

Other

Firdous Farooqui

M Parameswara Reddy

Maged A Michael

Naeem B Hanna

Meda Parameswara Reddy

Daniel A Keys

Parameswara Meda Reddy

Hong Li

Jackie S Bodnar

M Parameswara Reedy

M Paraweswara Reddy

MP Reddy

The present invention provides a useful system for assays that comprises a solid support, a plurality of capture oligonucleotides immobilized onto the solid support, and complementary oligonucleotides attached to capture ligands. A detectable label can be directly attached to the capture oligonucleotides or the complementary oligonucleotides. The labeled oligonucleotides can be detected, and used to determine the quality of the assay. A labeled detector ligand corresponding to a target ligand can also be independently detected apart from the labeled oligonucleotide.

Assay system using labeled oligonucleotides

This invention provides dye phosphoramidites, particularly phosphoramidites of substituted cyclic bridged cyanine and related dyes, of the general formula:  In this formula, each dotted line represents carbon atoms necessary to form a fused substituted or unsubstituted aromatic ring; m is an integer from 1 to 18; Y and Z are independently selected from the group consisting of S, O, N, CHand C(CH); Ris an alkyl; (PAM) is a phosphoramidite group; Xis a negative ion; and Q is L-W, wherein L is a conjugated cyclic moiety and W is OR, wherein Ris a second alkyl. Methods of making and using the dye phosphoramidites are also provided.

Stable cyanine dye phosphoramidites

The invention is an oligonucleotide-based assay system and kit useful for sorting, detecting, and identifying analytes. The system utilizes complementary oligonucleotide pairs in which one oligonucleotide of each pair is immobilized to a solid substrate and the other oligonucleotide has an analyte binding agent attached to it. The different oligonucleotide pairs hybridize at substantially the same rate, have substantially the same Tm, have nucleotide sequences designed to minimize cross-hybridization between different pairs, and hybridize together relatively rapidly at ambient temperatures without detectable cross-hybridization.

Oligonucleotide pairs for multiplexed binding assays

The present invention relates to an improved method for forming a protein-oligonucleotide conjugate. The method is particularly amenable for forming antibody-oligonucleotide conjugates. The invention further concerns the conjugate molecules produced using such improved methods.

Efficient synthesis of protein-oligonucleotide conjugates

Oligonucleotide probes, kits, and methods useful for detecting a polynucleotide target in a sample are provided. The method, a mixture is formed by combining a polynucleotide target sample, a first probe that is complementary to the polynucleotide target and having a first fluorescent donor or fluorescent acceptor; and a second probe that is partially complementary to the first probe and having a second fluorescent donor or fluorescent acceptor. The second probe competes with the polynucleotide target for binding to the first probe and the first probe preferentially binds to the polynucleotide target rather than to the second probe. The-first fluorescent donor or acceptor and second fluorescent donor or acceptor form a donor/acceptor pair capable of fluorescence resonance energy transfer (FRET) with each other in response to activation of the fluorescent donor by light of a predetermined wavelength or band of wavelengths.

Oligonucleotide probes for detecting nucleic acids through changes in flourescence resonance energy transfer

Efficient cyclic-bridged cyanine dyes

This invention provides dye phosphoramidites, particularly phosphoramidites of cyanine and related dyes, of the general formula:

Cyanine dye phosphoramidites

Activating groups based on N-hydroxynaphthalimide, are disclosed herein. The activating groups can mediate the coupling of labeling moieties, such as biotin or cyanine dyes, to a variety of components, including chain terminators, nucleoside triphosphates, and oligonucleotides, which are used in nucleotide sequencing. From these activating groups, activated esters of the labeling moieties can be prepared. The activated esters react with a component, for example a derivatized nucleotide chain terminator, to give a labeled component. In additions, methods of the present invention provide for labeling a nucleoside triphosphate in organic media. The activating groups and methods of the present invention allow the activation and coupling reactions to occur at a much higher yield, compared with the prior art.

Efficient activated cyanine dyes

Universal solid support oligonucleotide synthesis reagents, oligonucleotide synthesis processes, and reagents for cleaving oligonucleotides from solid supports are disclosed. Oligonucleotide synthesis reagents have the following general formula:

Universal solid supports and methods for their use

A compound of the general formula ##STR1## wherein R is alkyl of 1 to about 10 carbons and R' is selected from the group consisting of trityl and pixyl, for use in the synthesis of 2'-OMe RNA sequences. Fast cleavage and deprotection of oligonucleotides is facilitated by the use of a reagent comprising methylamine as active component in place of the traditional reagent ammonium hydroxide.

2'-OMe C.sup.AC phosphoramidite and methods for preparation and use

Oxidizing compositions particularly for use in automated oligonucleotide synthesis containing a mixture of KI and I.sub.2 in solution, in equilibrium with KI.sub.3. One preferred composition contains 1.75% KI.sub.3 (providing 0.69% KI and 1.06% I.sub.2) in tetrahydrofuran/pyridine/water (93/5/2, v/v). These formulations enable synthesis of oligonucleotides of significantly higher quality than the currently employed formulation comprising 3% I.sub.2 in tetrahydrofuran/pyridine/water (74/21/2, v/v).

Oxidizing reagent for use in oligonucleotide synthesis

Purification of synthesized oligonucleotides is achieved using novel processes and reagents. The novel processes involve adding certain salts to treated synthesized oligonucleotides prior to eluting the oligonucleotides from reverse phase purification cartridges. The novel processes also involve treating synthesized oligonucleotides using cleaving and deprotecting reagents containing methylamine and certain salts. Reagents comprising methylamine and certain salts can be used to rapidly cleave and deprotect oligonucleotides while obtaining high yields of purified oligonucleotides using reverse phase purification technologies.

Processes for purifying synthetic oligonucleotides

A compound of general formula ##STR1## wherein R is methyl R' is selected from the group consisting of trityl and pixyl, and R' is H or OMe. These compounds may advantageously be employed in the synthesis of oligonucleotides by conventional methods, such as automated solid phase synthesis. Use of ethylene diamine in the cleavage and deprotection procedure substantially eliminates the formation of undesirable side products.

C.sup.ac -methylphosphonamidites and methods for preparing

Disclosed herein are N.sup.4 protected deoxycytidines for use in the synthesis of oligonucleotides, the protecting groups being represented by the formula: --CO--(CH.sub.2).sub.0-9 --CH.sub.3. Preferred embodiments are N.sup.4 acetyl deoxycytidines and include N.sup.4 acetyl deoxycytidine phosphoramidites and N.sup.4 acetyl deoxycytidine H-phosphonates. When used to prepare oligonucleotides the protected deoxycytidine compounds provide high quality oligonucleotide products with little side product from cleavage and deprotection reactions carried out with alkyl amine compounds.

CAC H-phosphonate and its use in the synthesis of oligonucleotides

Deoxyribonucleotide and ribonucleotide derivatives of the general formula I ##STR1## wherein R.sup.1 represents --CR'R'--Ar, in which Ar is substituted aryl (as hereinafter defined) and R' and R' are independently selected from the group consisting of hydrogen and lower alkyl; one of --R.sup.2 and R.sup.3 is a hydroxyl-protecting group and the other is a group suitable for synthesis of polynucleotides or for attachment of the nucleotide to a solid support; R.sup.4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base. When synthesis is carried out using these derivatives, the deprotection procedure is reduced to an essentially instantaneous process. The derivatives have acceptable shelf life and are very stable to conventional DNA synthesis conditions. Particularly preferred are those compounds wherein Ar is mono- and dihalo-substituted phenyl.

Growing community of inventors

Brea, CA, United States of America

Firdous Farooqui