Methods and compositions 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 a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, a cleaving agent scavenger comprising a disulfide bond, and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label attached through a cleavable disulfide linker and contains a removable chemical moiety capping the 3′
-OH group;
b) incorporating a first nucleotide analogue with said polymerase;
c) detecting the label of the incorporated nucleotide analogue;
d) removing the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group with said cleaving agent; and
e) incorporating a second nucleotide analogue in the presence of said cleaving agent scavenger.
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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
31 Claims
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1. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, a cleaving agent scavenger comprising a disulfide bond, and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label attached through a cleavable disulfide linker and contains a removable chemical moiety capping the 3′
-OH group;b) incorporating a first nucleotide analogue with said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) removing the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group with said cleaving agent; ande) incorporating a second nucleotide analogue in the presence of said cleaving agent scavenger. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, a cleaving agent scavenger comprising an azido group, 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;b) incorporating a first nucleotide analogue with said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) removing the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group and cleaving the cleavable linker with said cleaving agent; ande) incorporating a second nucleotide analogue in the presence of said cleaving agent scavenger. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, cystamine, and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label, said label attached by a cleavable linker comprising a disulfide bond; b) incorporating a first nucleotide analogue with said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) removing the label of the incorporated nucleotide analogue by cleaving the cleavable linker with said cleaving agent; and e) incorporating a second nucleotide analogue in the presence of said cystamine. - View Dependent Claims (24, 25, 26, 27, 28)
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29. A method of incorporating labeled nucleotides into nucleic acid, comprising:
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a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, 11-Azido-3,6,9-trioxaundecan-1-amine, 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;b) incorporating a first nucleotide analogue with said polymerase; c) detecting the label of the incorporated nucleotide analogue; d) removing the chemical moiety of the incorporated nucleotide analogue capping the 3′
-OH group with said cleaving agent; ande) incorporating a second nucleotide analogue in the presence of said 11-Azido-3,6,9-trioxaundecan-1-amine. - View Dependent Claims (30, 31)
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Specification