Conformational probes and methods for sequencing nucleic acids
First Claim
1. A method of determining a sequence of nucleotides for a nucleic acid sample, comprisingproviding an array of nucleic acid templates, wherein the nucleic acid templates comprise nucleotide sequence fragments of the nucleic acid sample;
- contacting the array of nucleic acid templates with conformationally labeled polymerases and at least four different nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates, wherein the different nucleotide species comprise at least one non-natural nucleotide analog, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species;
detecting a series of the conformational signal changes from the conformationally labeled polymerase under the conditions and at individual locations of the array; and
determining the sequence of nucleotides for the nucleic acid sample by determining the rates or time durations for the conformational signal changes at the individual locations of the array, wherein the determining comprises distinguishing at least 10 fold difference between the rate or time duration for the conformational signal changes of the at least one non-natural nucleotide analog and the other different nucleotide species that are added to form the nucleic acid complements of the nucleic acid templates, and wherein the at least one non-natural nucleotide analog lacks probes that are detected in the method to determine the sequence of nucleotides for the nucleic acid sample.
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Abstract
This disclosure provides a method of determining a sequence of nucleotides for a nucleic acid template. The method can include the steps of contacting the nucleic acid template with a conformationally labeled polymerase and at least four different nucleotide species under conditions wherein the conformationally labeled polymerase catalyzes sequential addition of the nucleotide species to form a nucleic acid complement of the nucleic acid template, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions; and determining the rates or time durations for the changes in the signal, thereby determining the sequence of nucleotides for the nucleic acid template.
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Citations
41 Claims
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1. A method of determining a sequence of nucleotides for a nucleic acid sample, comprising
providing an array of nucleic acid templates, wherein the nucleic acid templates comprise nucleotide sequence fragments of the nucleic acid sample; -
contacting the array of nucleic acid templates with conformationally labeled polymerases and at least four different nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates, wherein the different nucleotide species comprise at least one non-natural nucleotide analog, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of the conformational signal changes from the conformationally labeled polymerase under the conditions and at individual locations of the array; and determining the sequence of nucleotides for the nucleic acid sample by determining the rates or time durations for the conformational signal changes at the individual locations of the array, wherein the determining comprises distinguishing at least 10 fold difference between the rate or time duration for the conformational signal changes of the at least one non-natural nucleotide analog and the other different nucleotide species that are added to form the nucleic acid complements of the nucleic acid templates, and wherein the at least one non-natural nucleotide analog lacks probes that are detected in the method to determine the sequence of nucleotides for the nucleic acid sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 23, 24, 25)
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16. A method of determining nucleotide sequences, comprising
(a) providing an array of different nucleic acid templates; -
(b) providing a mixture of nucleotide species, the mixture comprising (i) at least four different nucleotide species, (ii) at least one of the four different nucleotide species comprising a reversible terminator moiety, and (iii) at least two of the four different nucleotide species comprising an extendible 3′
hydroxyl moiety;(c) contacting the array of nucleic acid templates with conformationally labeled polymerases and the mixture of nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase, wherein the rate or time duration for the conformational signal change is distinguishable for the at least two nucleotide species comprising the extendible 3′
hydroxyl moiety, andwherein a plurality of the nucleic acid complements incorporate the at least one nucleotide species that comprises the reversible terminator moiety; (d) removing the reversible terminator moiety; (e) detecting a series of the conformational signal changes from the conformationally labeled polymerase at individual locations of the array; and (f) determining the sequence of nucleotides for the nucleic acid sample from the series of the conformational signal changes in the signal from the conformationally labeled polymerase, wherein the rate or time duration for the conformational signal changes are distinguished for the at least two nucleotide species comprising the extendible 3′
hydroxyl moiety that are added to form the nucleic acid complements of the nucleic acid templates. - View Dependent Claims (17, 22, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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Specification