Real-time sequence determination
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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.
34 Citations
79 Claims
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34. A single molecule sequencing apparatus comprising:
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a reaction chamber including;
a polymerizing agent comprising a molecular fluorescent donor tag covalently bonded thereto and confined or immobilized within the reaction chamber, and a sequencing medium comprising an unknown nucleic acid template and a primer, where the primer is adapted to duplex to a portion of the template to form a primer-template complex and where the polymerizing agent and primer-template complex combine to form a sequencing complex, a first deoxynucleotide triphosphate (dNTP) feed chamber including a first tagged dNTP having a first molecular fluorescent acceptor tag covalently bonded thereto, a second deoxynucleotide triphosphate (dNTP) feed chamber including a second tagged dNTP having a second molecular fluorescent acceptor tag covalently bonded thereto, a third deoxynucleotide triphosphate (dNTP) feed chamber including a third dNTP, a fourth deoxynucleotide triphosphate (dNTP) feed chamber including a fourth dNTP, channels connecting the dNTP feed chambers to the reaction chamber, an excitation light source, and a fluorescent detector/analyzer, where the feed chambers are adapted to feed dNTPs to the reaction chamber, the light source is designed to irradiate the chamber with light of a wavelength sufficient to excite the donor tag on the polymerizing agent, and the detector/analyzer is designed to detect each fluorescent resonance energy transfer (FRET) event evidencing each incorporation of a tagged dNTP at the sequencing complex to produce a read out of each incorporation of a tagged dNTP. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
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57. A single molecule sequencing apparatus comprising:
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a reaction chamber including;
a substrate comprising a polymerizing agent comprising a molecular fluorescent donor tag covalently bonded thereto and immobilized on or to the substrate and, a sequencing medium comprising an unknown nucleic acid template and a primer, where the primer is adapted to duplex to a portion of the template to form a primer-template complex and where the polymerizing agent and primer-template complex combine to form a pre-sequencing complex, a first deoxynucleotide triphosphate (dNTP) feed chamber including a first tagged dNTP having a first molecular fluorescent acceptor tag covalently bonded thereto, a second deoxynucleotide triphosphate (dNTP) feed chamber including a second tagged dNTP having a second molecular fluorescent acceptor tag covalently bonded thereto, a third deoxynucleotide triphosphate (dNTP) feed chamber including a third dNTP, a fourth deoxynucleotide triphosphate (dNTP) feed chamber including a fourth dNTP, channels interconnecting the dNTP feed chambers with the reaction chamber, an excitation light source, and a fluorescent detector/analyzer, where the feed chambers are adapted to feed dNTPs to the reaction chamber, the light source is designed to irradiate the chamber with light of a wavelength sufficient to excite the donor tag on the polymerizing agent, and the detector/analyzer is designed to detect each fluorescent resonance energy transfer (FRET) event evidencing each incorporation of a tagged dNTP at the sequencing complex to produce a read out of each incorporation of a tagged dNTP. - View Dependent Claims (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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Specification