Pharmacia Biotech Inc.

Amersham Biosciences Ab

Pharmacia P-L Biochemicals Inc.

Motorola Corporation

Ge Healthcare Bio-Sciences Ab

Amersham Pharmacia Biotech, Inc.

Charles K Brush

Allyson J Beuhler

Robert E Lajos

Travis Johnson

John McGowen

Eric Dean Anderson

Robert Elghanian

Vineet Gupta

Abhijit Mazumder

W Travis Johnson

R Scott Duthie

Chang-Gong Liu

Yanzheng Xu

Ned D Reimer

Eugene P Stirchak

Mark E Freeman

Lawrence J Burazin

An assay is provided for nucleic acids that can be post-synthetically labeled, wherein modified nucleoside triphosphates are used that are more efficiently and specifically incorporated during nucleic acid synthesis than labeled nucleoside triphosphates. In a preferred embodiment, nucleoside &#x3b1;-thiotriphosphates are utilized. Maleimide or iodoacetamide conjugating moieties can be attached post-synthetically. The conjugating moieties may include a reporter group. Also disclosed are new methods for detecting single nucleotide polymorphism.

Post-synthesis labeling of nucleic acids, assays using nucleic acids that are labeled post-synthetically, single nucleotide polymorphism detection, and associated compounds and microarrays

Methods and devices for detecting nucleic acid and protein targets on hydrogel microarrays are disclosed. Fluorophores are incorporated into the targets and detected. A linear correlation between target concentration and signal amplitude is maintained through the elimination of active enzyme amplification.

Hydrogel-based microarray signal amplification methods and devices therefor

The present invention provides solid supports (e.g., glass) and polymer hydrogels (particularly polymer hydrogel arrays present on a solid support) comprising one or more reactive sites for the attachment of biomolecules, as well as biomolecules comprising one or more reactive sites for attachment to solid supports and polymer hydrogels. The invention further provides novel compositions and methods for the preparation of biomolecules, solid supports, and polymer hydrogels comprising reactive sites. The invention also provides for preparation of crosslinked solid supports, polymer hydrogels, and hydrogel arrays, wherein one or more biomolecules is attached by means of the reactive sites in a photocycloaddition reaction. Advantageously, according to the invention, crosslinking of the hydrogel and attachment of biomolecules can be done in a single step.

Methods and compositions for attachment of biomolecules to solid supports, hydrogels, and hydrogel arrays

&lsqb;2&plus;2&rsqb; photo-attachable functional groups were incorporated in polyacrylamide based hydrogels and synthetic oligonucleotide probes. The probes were photochemically attached by covalent bonding to the three dimensional surface of a hydrogel.

Use and evaluation of a &lsqb;2&plus;2&rsqb; photoaddition in immobilization of oligonucleotides on a three-dimensional hydrogel matrix

A chemical compound of the following formula: ##STR1## wherein R.sup.1 is selected from the group consisting of alkyl, aralkyl, and substituted alkyl groups; R.sup.3 is selected from the group consisting of H, PO.sub.3.sup.-2, P.sub.2 O.sub.6.sup.-3 ; P.sub.3 O.sub.9.sup.-4, and .alpha.-thio phoshates (PSO.sub.2.sup.-2 ; P.sub.2 SO.sub.5.sup.-3 ; P.sub.3 O.sub.8.sup.-4); and .alpha.BH.sub.3.sup.- phosphates (P(BH.sub.3)O.sub.2.sup.-2, P.sub.2 (BH.sub.3)O.sub.5.sup.-3, P.sub.3 (BH.sub.3)O.sub.8.sup.-4); R.sup.4 is selected from the group consisting of H, lower alkyl, acyl, (CH.sub.2).sub.p COO(CH.sub.2).sub.q CH.sub.3 wherein p is an integer from 0 to 4 and q is an integer from 0 to 4, and 5,6; 6,7; or 7,8-butadienyl; R.sup.5 is selected from the group consisting of H lower alkyl, acyl, (CH.sub.2).sub.p COO(CH.sub.2).sub.q CH.sub.3 wherein p is an integer from 0 to 4 and q is an integer from 0 to 4 and 5,6; 6,7; or 7,8- butadienyl; r is 1, 2, or 3 to form a second fused aromatic; X or Y are selected from the group consisting of O, S, C(R.sup.6).sub.2, or N(R.sub.6), wherein R.sup.6 is preferably CH.sub.3 or a lower alkyl; and R.sup.3 --O-Sugar-Base is a nucleoside or nucleotide is disclosed.

Non-sulfonated cyanine dyes for labeling nucleosides and nucleotides

A preparation of a labelled nucleotide comprising at least one compound having a Mg.sup.2+ association constant between 1.times.10.sup.-11 to 1.times.10.sup.-2, inclusive. The compound is preferably selected from the group consisting of citrate, isocitrate, phosphate, EGTA, EDTA, and CDTA. The concentration of the compound is preferably at least 5 mM.

Composition for stabilization of labelled nucleoside triphosphates and

A chemical compound of the following formula is disclosed: ##STR1## where R is selected from the group consisting of H, trityl, 4-O-monomethoxytrityl, 4,4'-O-dimethoxytrityl, and acyl groups, and whereby R can be used as a protecting group or is an H;

Indocarbocyanine and benzindocarbocyanine phosphoramidites

Compounds useful to attach a fluorescein label to an oligonucleotide are disclosed. To create these compounds, oxygen groups on the fluorescein moiety are protected (e.g. with acyl groups). One then links a phosphoramidite to the fluorescein moiety via an amide or thiourea linkage to an active site on the fluorescein ring. The resulting compound can then be directly linked to an oligonucleotide as it is being formed in an automated synthesizer and then deprotected using the same deprotection conditions as are used to deprotect the oligonucleotides. Such compounds are, inter alia, useful to create labelled primers for DNA sequencing.

Method of attaching a fluorescent label to an oligonucleotide

Fluorescein labelled phosphoramidites

A chemical compound of the following formula is disclosed ##STR1## where R is one of H, trityl, 4-0-monomethoxytrityl, 4,4'-O-dimethoxytrityl, or acyl groups and R may be used as a protecting group or is an H; R.sup.1 is a phosphoramidite; R.sup.11 is one of H or lower alkyl groups; R.sup.111 is one of H or lower alkyl groups; R.sup.4 is one of H, lower alkyl, acyl, or (CH.sub.2).sub.p COO(CH.sub.2).sub.q CH.sub.3 where p is an integer from 0 to 4 and q is an integer from 0 to 4; R.sup.5 is one of H, lower alkyl, acyl, or (CH.sub.2).sub.p COO(CH.sub.2).sub.q CH.sub.3 where p is an integer from 0 to 4 and q is an integer from 0 to 4; n is an integer from 0 to 10; m is an integer from 0 to 10; and r is 1, 2, or 3.

Indocarbocyanine-linked phosphoramidites

Ether variant lipids bound to phosphoramidites are disclosed. These can be used to create oligonucleotides that are linked to stable lipid-like units. For example, dialkyl glycerols are linked to phosphoramidites.

Lipo-phosphoramidites

Compounds useful for attaching a fluorescein label to an oligonucleotide are disclosed. The compounds are fluorescein-linked phosphoramidites where the hydroxyl groups on the fluorescein moiety are protected by isobutyryl groups. An example of this class of compound is ##STR1##

Growing community of inventors

Whitefish Bay, WI, United States of America

Charles K Brush