Patent ID: 7462468

Claim:
A method for detecting incorporation of at least one nucleotide phosphate into a single primer nucleic acid molecule, said method comprising: i. immobilizing onto a support a polymerase nucleic acid complex comprising a target nucleic acid, a primer nucleic acid which complements a region of the target nucleic acid, and at least one nucleic acid polymerase; ii. contacting said immobilized complex with at least one type of labeled nucleotide phosphate of the formula II: wherein: Dye 1 is selected from the group consisting of TO-1, TO-3, BO-1, BO-3, YO-1, YO-3, JO-1, JO-3, PO-1, PO-3, LO-1, LO-3, YO-1, YO-3, propidium and psoralen; Dye 2 is selected from the group consisting of TO-1, TO-3, BO-1, BO-3, YO-1, YO-3, JO-1, JO-3, PO-1, PO-3, LO-1, LO-3, YO-1, YO-3, propidium and psoralen; r is 1-6; t is 1-6; n is 0-20; Z is a member selected from the group consisting of O, S, CR 1 R 2 , NR 3 R 4 , and P(O) 2 ; R 1 , R 2 , R 3 , and R 4 are each members independently selected from the group consisting of H and optionally substituted alkyl; L is a member selected from the group consisting of a direct link, or a covalent linkage, wherein said covalent linkage is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-100 atoms selected from the group consisting of C, N, P, O, and S, wherein L can have additional hydrogen atoms to fill valences, and wherein said linkage contains any combination of ether, thioether, amine, ester, carbamate, urea, thiourea, oxy or amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen, or nitrogen-platinum bonds; or aromatic or heteroaromatic bonds; and NP is a nucleotide phosphate [NP]; and iii. detecting the incorporation of said at least one type of labeled NP into a single molecule of said primer, while said at least one type of labeled NP is in contact with said immobilized complex, by detecting the label of the NP while said at least one type of labeled NP is in contact with said polymerase nucleic acid complex.