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US 2014/0274732 A1Published application
Methods and compositions for nucleic acid sequencing using electronic sensing elements
AssigneePacific Biosciences of California, Inc.
InventorsJeremiah HANES, Jonas KORLACH, Stephen TURNER
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Abstract
The present invention is directed to methods, devices, compositions and systems for obtaining sequence data from nucleic acid templates by utilizing electronic sensing elements.
Claims61
A method of identifying a sequence of a plurality of template nucleic acids, said method comprising:
- (a) providing a plurality of immobilized clonal populations of primed nucleic acid templates, each clonal population proximate to an electronic sensing element;
- (b) exposing the plurality of immobilized clonal populations to a first type of nucleoside polyphosphate under conditions supporting a template directed incorporation of a nucleoside monophosphate portion of the first type of nucleoside polyphosphate;
- wherein the first type of nucleoside polyphosphate comprises a polyphosphate chain of three or more phosphates and a terminal blocking group, and wherein the incorporation reaction is carried out in the presence of a phosphatase enzyme and results in the cleavage of an alpha-beta phosphate bond and at least one additional phosphate bond of the incorporated nucleoside polyphosphate;
- (c) electrically monitoring each of the clonal populations with the electronic sensing elements to detect whether one or more incorporations of the first type of nucleoside polyphosphate occurs at that clonal population;
- (d) repeating steps (b) and (c) with second, third and fourth types of nucleoside polyphosphates,
- wherein said repeating step (d) is conducted a number of times to thereby identify the sequence of the plurality of template nucleic acids.
3 more elements
A method of identifying a sequence of a plurality of template nucleic acids, said method comprising:
- (a) providing a plurality of single-molecule polymerase-template complexes, each complex comprising a template nucleic acid, a polymerase enzyme and a primer;
- wherein each complex is associated with an electronic sensing element;
- (b) exposing the complexes to two or more types of nucleoside polyphosphates, wherein the two or more types of nucleoside polyphosphates each comprises a phosphate chain of three or more phosphates and a terminal blocking group, and wherein each type of nucleoside polyphosphate has a different number of phosphates;
- the exposing carried out under conditions supporting template dependent primer extension through multiple incorporation reactions, whereby the incorporation reactions extending the primer are carried out in the presence of a phosphatase enzyme resulting in the cleavage of an alpha-beta phosphate bond and at least one additional phosphate bond of the incorporated nucleoside polyphosphates;
- and
- (c) detecting the phosphate bond cleavages resulting from the incorporation reactions with the electronic sensing elements to identify the types of nucleoside polyphosphates incorporated in the incorporation reactions to thereby sequence the plurality of template nucleic acids.
3 more elements
A method of identifying a sequence of a plurality of template nucleic acids, said method comprising:
- (a) providing a plurality of immobilized single-molecule primed nucleic acid templates, wherein each single molecule template is proximate to an electronic sensing element;
- (b) exposing the plurality of immobilized single molecules to a first type of nucleoside polyphosphate under conditions supporting a template directed incorporation of a nucleoside monophosphate portion of the first type of nucleoside polyphosphate and in the presence of a phosphatase enzyme;
- wherein the first type of nucleoside polyphosphate comprises a polyphosphate chain of three or more phosphates and a terminal blocking group;
- and whereby, upon incorporation, cleavage of the alpha-beta phosphate bond and cleavage of at least one additional phosphate bond of the polyphosphate chain occurs;
- (c) electrically monitoring each of the single molecule templates with the electronic sensing elements to detect whether one or more incorporations of the type of nucleoside polyphosphate occurs at that single-molecule template;
- (d) repeating steps (b) and (c) with second, third and fourth types of nucleoside phosphates,
- wherein said repeating step (d) is conducted a number of times to thereby identify the sequence of the plurality of template nucleic acids.
4 more elements
A method for increasing a signal from a template directed incorporation of a nucleoside monophosphate portion of a nucleoside polyphosphate, the method comprising:
- (a) providing a plurality of immobilized clonal populations of primed nucleic acid templates, each clonal population proximate to an electronic sensing element;
- (b) exposing the plurality of immobilized clonal populations to a first type of nucleoside polyphosphate under conditions supporting a template directed incorporation of a nucleoside monophosphate portion of the first type of nucleoside polyphosphate;
- wherein the first type of nucleoside polyphosphate comprises a polyphosphate chain of three or more phosphates and a terminal blocking group;
- and whereby, upon incorporation, cleavage of the alpha-beta phosphate bond and cleavage of at least one additional phosphate bond of the polyphosphate chain occurs, thereby generating a signal detectable by the electronic sensing elements;
- (c) electrically monitoring each of the clonal populations with the electronic sensing elements to detect whether one or more incorporations of the type of nucleoside polyphosphate occurs at that clonal population by detecting the signal generated by cleavage of the alpha-beta phosphate bond and the at least one additional phosphate bond;
- (d) repeating steps (b) and (c) with second, third and fourth types of nucleoside phosphates,
- wherein the repeating step (d) is conducted a number of times to thereby identify the sequence of the plurality of template nucleic acids.
4 more elements
A method for increasing a signal from a template directed incorporation of a nucleoside monophosphate portion of a nucleoside polyphosphate, the method comprising:
- (a) providing a plurality of single-molecule polymerase-template complexes, each complex comprising a template nucleic acid, a polymerase enzyme and a primer;
- wherein each complex is associated with an electronic sensing element;
- (b) exposing the complexes to two or more types of nucleoside polyphosphates, wherein the two or more types of nucleoside polyphosphates each comprises a phosphate chain of three or more phosphates, and wherein each type of nucleoside polyphosphate has a different number of phosphates and a terminal blocking group;
- the exposing carried out under conditions supporting template dependent primer extension through multiple incorporation reactions, whereby the incorporation reactions extending the primer are carried out in the presence of a phosphatase enzyme resulting in the cleavage of an alpha-beta phosphate bond and at least one additional phosphate bond of the incorporated nucleoside polyphosphates, thereby generating a signal detectable by the electronic sensing elements;
- and
- (c) detecting the signals from the phosphate bond cleavages resulting from the incorporation reactions with the electronic sensing elements to identify the types of nucleoside polyphosphates incorporated in the incorporation reactions to thereby sequence the plurality of template nucleic acids.
3 more elements
A method for identifying a sequence of a plurality of template nucleic acids, said method comprising:
- (a) providing a plurality of immobilized clonal populations of nucleic acids, wherein each clonal population is proximate to an electronic sensing element;
- (b) exposing the plurality of immobilized clonal populations to a first type of nucleoside polyphosphate under conditions supporting a template directed incorporation of a nucleoside monophosphate portion of the first type of nucleoside polyphosphates into primers hybridized to the nucleic acids;
- wherein the first type of nucleoside polyphosphate comprises a polyphosphate chain of three or more phosphates and a terminal blocking group;
- and whereby, upon incorporation, cleavage of the alpha-beta phosphate bond and cleavage of at least one additional phosphate bond of the polyphosphate chain occurs, thereby releasing at least three hydrogen ions;
- (c) electrically monitoring each of the clonal populations with the electronic sensing elements to detect whether one or more incorporations of the first type of nucleoside polyphosphate occurs at that clonal population by detecting the released hydrogen ions at that clonal population;
- (d) repeating steps (b) and (c) with second, third and fourth types of nucleoside phosphates,
- wherein the repeating step (d) is conducted a number of times to thereby identify the sequence of the plurality of template nucleic acids.
4 more elements
A method for identifying a sequence of a plurality of template nucleic acids, the method comprising:
- (a) providing a plurality of immobilized clonal populations of primed nucleic acid templates, each clonal population proximate to an electronic sensing element;
- (b) exposing the plurality of immobilized clonal populations to a first type of nucleoside polyphosphate under conditions supporting a template directed incorporation of a nucleoside monophosphate portion of the first type of nucleoside polyphosphate;
- wherein the first type of nucleoside polyphosphate comprises a polyphosphate chain of three or more phosphates and a terminal blocking group;
- and whereby, upon incorporation, cleavage of the alpha-beta phosphate bond and cleavage of at least one additional phosphate bond of the polyphosphate chain occurs, thereby generating a byproduct detectable by the electronic sensing element;
- (c) electrically monitoring each of the clonal populations with the electronic sensing elements to detect whether one or more incorporations of the type of nucleoside polyphosphate occurs at that clonal population by detecting the byproduct generated by the cleavage of the phosphate bonds;
- (d) repeating steps (b) and (c) with second, third and fourth types of nucleoside phosphates,
- wherein the repeating step (d) is conducted a number of times to thereby identify the sequence of the plurality of template nucleic acids.
4 more elements
Dependencies are inferred from the claim text.