Reagents containing terminal-phosphate-labeled nucleotides for nucleic acid sequencing
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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 arid the sequence of the target nucleic acid is determined.
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Citations
77 Claims
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1-61. -61. (canceled)
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62. A nucleic acid sequencing reagent comprising:
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at least one or more terminal-phosphate-labeled nucleotides or nucleotide analogs having the formula;
Base unit—
sugar moiety —
Y—
(P)n—
P—
label moiety;
wherein the base unit is selected from the group consisting of adenosine, thymidine, cytidine and guanosine base unit, wherein P is a phosphate group and n is an integer of at least 2, and wherein Y is an oxygen or sulfur atom, wherein the reagent is present in a sufficient quantity for performing a sequencing reaction by nucleic acid polymerization. - View Dependent Claims (63, 64, 65, 66, 67, 68, 77)
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69. A nucleic acid sequencing combination comprising:
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(a) at least one or more terminal-phosphate-labeled nucleotides or nucleotide analogs having the formula;
Base unit—
Sugar moiety —
Y—
(P)n—
P—
Label moietywherein the base unit is selected from the group consisting of adenosine, thymidine, cytidine and guanosine base unit, wherein P is a phosphate group and n is an integer of at least 2, wherein Y is an oxygen or sulfur atom; and
(b) instructions for performing the nucleic acid sequencing. - View Dependent Claims (70, 71, 72, 73, 74, 75, 76)
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Specification