METHODS FOR SEQUENCE DETERMINATION
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.
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, 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.
2 . The system of claim 1 , wherein the complex does not comprise a quencher.
3 . The system of claim 1 , wherein the detector further comprises data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels.
4 . The system of claim 1 , further comprising a plurality of active complexes confined within the viewing field and where the detector further comprises data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels and where the active complexes are distributed within the viewing field to minimize data channels having a multiple active complexes therein.
5 . The system of claim 1 , wherein the monomer types comprise tags and where the tags on each monomer type are all different and where the detectable properties are fluorescence from each tag.
6 . The system of claim 5 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.
7 . The system of claim 6 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tags are brought into close proximity of between 100 Å and 10 Å.
8 . An apparatus for determining sequence information at the single molecule level comprising:
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.
9 . The apparatus of claim 8 , wherein the complex does not comprise a quencher.
10 . The apparatus of claim 8 , wherein the detector further comprises data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels.
11 . The apparatus of claim 8 , further comprising a plurality of active complexes confined within the viewing field and where the detector further comprises data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels and where the active complexes are distributed within the viewing field to minimize data channels having a multiple active complexes therein.
12 . The apparatus of claim 8 , wherein the monomer types comprise tags and where the tags on each monomer type are all different and where the detectable properties are fluorescence from each tag.
13 . The apparatus of claim 12 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.
14 . The apparatus of claim 13 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tags are brought into close proximity of between 100 Å and 10 Å.
15 . A method for determining sequence information at the single molecule level comprising:
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.
16 . The method of claim 15 , wherein the complex does not comprise a quencher.
17 . The method of claim 15 , wherein the detector further comprises data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels.
18 . The method of claim 15 , further comprising a plurality of active complexes confined within the viewing field and where the detector further comprises data channels within the viewing field, each data channel comprising one pixel or a plurality of pixels and where the active complexes are distributed within the viewing field to minimize data channels having a multiple active complexes therein.
19 . The method of claim 15 , wherein the monomer types comprise tags and where the tags on each monomer type are all different and where the detectable properties are fluorescence from each tag.
20 . The method of claim 19 , wherein the polymerizing agent tag is a donor fluorophore and the monomer tags are acceptor fluorophores, each monomer type having a different fluorophore having a different fluorescent signature and the acceptors fluoresce after fluorescent resonance energy transfer (FRET) with the donor.
21 . The method of claim 20 , wherein the tag on the polymerizing agent is positioned so that during monomer incorporation, the donor and acceptor tags are brought into close proximity of between 100 Å and 10 Å.