Methods and solutions for inhibiting undesired cleaving of labels
First Claim
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1. A method of incorporating labeled nucleotides into nucleic acid, comprising:
- a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′
-OH group, iii) a second solution comprising a cleaving agent, iv) a cleaving agent scavenger, wherein said cleaving agent scavenger is cystamine, and v) a wash solution;
b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase;
c) detecting the label of the incorporated nucleotide analogue;
d) introducing said second solution into said reaction chamber under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group is removed by said cleaving agent leaving a 3′
hydroxyl group to enable the next nucleotide incorporation;
e) washing said reaction chamber with said wash solution under conditions where some leftover cleaving agent remains in said reaction chamber; and
f) introducing said cleaving agent scavenger into said reaction chamber under conditions where cystamine reacts with said leftover cleaving agent.
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Abstract
The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.
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Citations
39 Claims
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1. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′
-OH group, iii) a second solution comprising a cleaving agent, iv) a cleaving agent scavenger, wherein said cleaving agent scavenger is cystamine, and v) a wash solution;b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) introducing said second solution into said reaction chamber under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group is removed by said cleaving agent leaving a 3′
hydroxyl group to enable the next nucleotide incorporation;e) washing said reaction chamber with said wash solution under conditions where some leftover cleaving agent remains in said reaction chamber; and f) introducing said cleaving agent scavenger into said reaction chamber under conditions where cystamine reacts with said leftover cleaving agent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues selected from the group consisting of cytosine, thymine, deaza-adenine and deaza-guanine, wherein each nucleotide analogue comprises a unique label attached through a cleavable linker to a 5-position of cytosine or thymine or to a 7-position of deaza-adenine or deaza-guanine, and wherein each nucleotide analogue contains a removable chemical moiety capping the 3′
-OH group, iii) a second solution comprising a cleaving agent, iv) a cleaving agent scavenger, wherein the cleaving agent scavenger is cystamine, and v) a wash solution;b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) introducing said second solution into said reaction chamber under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group is removed leaving a 3′
hydroxyl group to enable the next nucleotide incorporation, and said cleavable linker is cleaved by said cleaving agent;e) washing said reaction chamber with said wash solution under conditions where some leftover cleaving agent remains in said reaction chamber; and f) introducing said cleaving agent scavenger into said reaction chamber under conditions where cystamine reacts with said leftover cleaving agent. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label, said label attached via a cleavable linker, wherein said cleavable linker comprises a disulfide bond, iii) a second solution comprising a cleaving agent, iv) a cleaving agent scavenger, wherein the cleaving agent scavenger comprises a disulfide bond, and v) a wash solution; b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) introducing said second solution into said reaction chamber under conditions such that the label of the incorporated nucleotide analogue is removed by cleaving said cleavable linker with said cleaving agent; e) washing said reaction chamber with said wash solution under conditions where some leftover cleaving agent remains in said reaction chamber; and f) introducing said cleaving agent scavenger into said reaction chamber under conditions wherein said scavenger reacts with said leftover cleaving agent. - View Dependent Claims (31, 32, 33, 34, 35)
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36. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′
-OH group, iii) a second solution comprising a cleaving agent, iv) a cleaving agent scavenger, wherein said cleaving agent scavenger is cystamine, and v) a wash solution;b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase; c) detecting the label of the incorporated nucleotide analogue so that the identity of the base can be called; d) introducing said second solution into said reaction chamber under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group is removed by said cleaving agent leaving a 3′
hydroxyl group to enable the next nucleotide incorporation;e) washing said reaction chamber with said wash solution under conditions where some leftover cleaving agent remains in said reaction chamber; and f) introducing said cleaving agent scavenger into said reaction chamber under conditions such that cystamine reacts with said leftover cleaving agent; and g) repeating steps b) through f) such that additional nucleotide analogs are incorporated, wherein each additional nucleotide analog is incorporated after a cleaving agent scavenger has been introduced into said reaction chamber, whereby the accuracy of base calling at step c) is improved. - View Dependent Claims (37)
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38. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′
-OH group, iii) a second solution comprising a cleaving agent, and iv) a wash solution comprising a cleaving agent scavenger;b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) introducing said second solution into said reaction chamber under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group is removed by said cleaving agent leaving a 3′
hydroxyl group to enable the next nucleotide incorporation; ande) washing said reaction chamber with said wash solution under conditions where leftover cleaving agent from step d) reacts with said scavenger. - View Dependent Claims (39)
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Specification