Sequence determination using multiply tagged polymerizing agents
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Abstract
A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.
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82 Claims
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- 20. A composition comprising a polymerase or reverse transcriptase including a pair of tags, an unknown nucleic acid template, a primer adapted of hybridize with a complementary sequence of a portion of the template to form a primer-template duplex, and monomers for the polymerase or reverse transcriptase, where a detectable property of at least one of the tags undergoes a change before, during and/or after each of a sequence of monomer incorporations.
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24. The composition of claim 22, wherein the polymerase comprises Taq DNA polymerase I having a at least one of the tags attached at a site selected from the group consisting of 513-518, 643, 647, 649 and 653-661.
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- 49. A composition comprising a polymerizing agent including at least one pair of tags, a nucleic acid template, a primer adapted to hybridize with a complementary sequence of the nucleic acid to form a primer-template duplex, and monomers for the polymerizing agent, where at least one of the tags in each pair has a detectable property that undergoes a change before, during and/or after each of a sequence of monomer incorporations.
- 66. A composition comprising a polymerizing agent including at least one pair of tags, a nucleic acid template, a primer adapted to hybridize with a complementary sequence of the nucleic acid to form a primer-template duplex, and monomers for the polymerizing agent, where the tags are substantially inactive when the polymerizing agent is in a first state and substantially active when the polymerizing agent is in a second state and where the polymerizing agent transition from the first state to the second state during each of a sequence of monomer incorporations.
Specification