Labeled nucleotide phosphate (NP) probes
First Claim
1. A method of identifying a nucleotide incorporated in a nascent strand in a nucleic acid polymerization reaction, comprising:
- providing an individual complex of a nucleic acid polymerase, a target polynucleotide and a primer that hybridizes to the target polynucleotide, positioned within a confined focal volume provided on a support;
introducing at least a first nucleotide or nucleotide analog bearing a first fluorescent label into the focal volume;
measuring a residence time of the at least first nucleotide or nucleotide analog within the focal volume, wherein the residence time of the at least first nucleotide or nucleotide analog is reflected by a period of duration of a fluorescence signal from the first fluorescent label in the focal volume, said period being longer than that of an unincorporated nucleotide or nucleotide analog randomly diffusing through the focal volume, thereby identifying a nucleotide incorporated by the polymerase of said individual complex in the nucleic acid polymerization reaction based upon the residence time of the nucleotide or nucleotide analog within said focal volume.
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Abstract
The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
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Citations
12 Claims
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1. A method of identifying a nucleotide incorporated in a nascent strand in a nucleic acid polymerization reaction, comprising:
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providing an individual complex of a nucleic acid polymerase, a target polynucleotide and a primer that hybridizes to the target polynucleotide, positioned within a confined focal volume provided on a support; introducing at least a first nucleotide or nucleotide analog bearing a first fluorescent label into the focal volume; measuring a residence time of the at least first nucleotide or nucleotide analog within the focal volume, wherein the residence time of the at least first nucleotide or nucleotide analog is reflected by a period of duration of a fluorescence signal from the first fluorescent label in the focal volume, said period being longer than that of an unincorporated nucleotide or nucleotide analog randomly diffusing through the focal volume, thereby identifying a nucleotide incorporated by the polymerase of said individual complex in the nucleic acid polymerization reaction based upon the residence time of the nucleotide or nucleotide analog within said focal volume. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of identifying a nucleotide incorporated in a nascent strand in a nucleic acid polymerization reaction, comprising:
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providing an individual complex of a nucleic acid polymerase, a target polynucleotide and a primer that hybridizes to the target polynucleotide, wherein the complex is positioned within a confined focal volume provided on a support; introducing at least a first labeled nucleotide or nucleotide analog to an active site of the nucleic acid polymerase; measuring a residence time of the nucleotide or nucleotide analog at the active site, thereby identifying a nucleotide or nucleotide analog incorporated by the polymerase of said individual complex based upon the length of the residence time of the incorporated nucleotide or nucleotide analog at the active site. - View Dependent Claims (8, 9, 10, 11)
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12. A method of identifying nucleotides incorporated in a primer extension reaction, comprising:
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providing an individual complex of a nucleic acid polymerase, a target polynucleotide and a primer that hybridizes to the target polynucleotide, in a confined focal volume provided on a support; allowing four different nucleotide and/or nucleotide analogs, each bearing a distinguishable fluorescent label, to freely diffuse into the focal volume; detecting the nucleotides and/or nucleotide analogs within the focal volume over time; identifying nucleotides and/or nucleotide analogs incorporated into a primer extension reaction by the individual complex based upon their residence time in the focal volume, wherein the length of the residence time of each nucleotide or nucleotide analog incorporated is longer relative to residence time for unincorporated nucleotides or nucleotide analogs.
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Specification