METHODS FOR SEQUENCE DETERMINATION
First Claim
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1. A system for determining sequence information at the single molecule level, comprising:
- a detector comprising a viewing field,at least one active complex confined within the viewing field, where the complex comprises;
a reaction buffer,a polymerizing agent,a template,a primer adapted to duplex with a portion of the template to form an extendable duplex, andmonomer types for the polymerizing agent,where the polymerizing agent and/or the monomer types comprising tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different; and
an excitation source adapted to excite the polymerizing agent and/or monomer tags within the viewing field,where the detector is adapted to detect changes in the detectable properties of the tags before, during and/or after one or a series of monomer incorporation events and to convert the incorporation event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template.
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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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Citations
21 Claims
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1. A system for determining sequence information at the single molecule level, comprising:
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a detector comprising a viewing field, at least one active complex confined within the viewing field, where the complex comprises; a reaction buffer, a polymerizing agent, a template, a primer adapted to duplex with a portion of the template to form an extendable duplex, and monomer types for the polymerizing agent, where the polymerizing agent and/or the monomer types comprising tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different; and an excitation source adapted to excite the polymerizing agent and/or monomer tags within the viewing field, where the detector is adapted to detect changes in the detectable properties of the tags before, during and/or after one or a series of monomer incorporation events and to convert the incorporation event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. An apparatus for determining sequence information at the single molecule level comprising:
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a substrate, a detector comprising a viewing field focused on a region of the substrate, at least one active sequencing complex confined within the viewing field and comprising a reaction buffer, a polymerizing agent, a template, a primer and monomer types for the polymerizing agent, where the polymerizing agent and/or the monomer types comprise tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different; and an excitation source adapted to excite the tags within the viewing field, where the detector is adapted to detect changes in the detectable properties of the tags before, during and/or after one monomer incorporation event or a series of monomer incorporation events and to convert the event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template. - View Dependent Claims (9, 10, 11, 12, 13, 14)
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15. A method for determining sequence information at the single molecule level comprising:
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providing a detector comprising a viewing field; providing at least one active sequencing complex confined within the viewing field, where the complex comprises; a reaction buffer, a polymerizing agent, a template, a primer adapted to duplex with a portion of the template to form an extendable duplex, and monomer types for the polymerizing agent, where the polymerizing agent and/or the monomer types comprise tags having detectable properties that undergo changes before, during and/or after a monomer incorporation event, where the tags and the detectable properties are same or different; an excitation source adapted to excite the tags within the viewing field, detecting changes in the detectable properties of the tags before, during and/or after one monomer incorporation event or a series of monomer incorporation events; and converting the event or events into an identity of one monomer or identities of a plurality of monomers complementary to a sequence of monomers in the template. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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Specification