Compositions and methods for 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.
137 Citations
40 Claims
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30. A system for determining sequence information from a single molecule comprising:
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a nucleic acid template;
a single-molecule sequencer comprising a polymerase including a polymerase tag, a primer adapted to hybridize to a portion of the template to form an extendable primer-template duplex and monomers for the polymerase, each monomer type including a different monomer tag, where at least one tag has a detectable property;
a excitation source adapted to excite the polymerase and/or monomer tags; and
a detector adapted to detect the detectable property, where the detectable property changes during polymerization of a complementary sequence of monomers and where the changes correspond to an identity of each nucleotide in the template. - View Dependent Claims (35)
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36. A system that enables detecting a signal corresponding to a detectable property evidencing changes in interactions between a synthesizing/polymerizing agent or a depolymerizing agent (molecule) and its substrates (monomers or depolymerizable polymers) and decoding the signal into monomer order specific information or monomer sequence information, preferably in real-time or near real-time.
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37. A system for determining sequence information from a single-molecule comprising a molecule having a sequence of known elements, a single-molecule sequencer comprising a polymerase having at least one tag associated therewith, a excitation source adapted to excite at least one tag on the polymerase, and a detector adapted to detect a response from the excited tag on the polymerase, where the response from at least one tag changes during polymerization of a complementary sequence of elements representing the element sequence of the molecule.
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38. A system for determining sequence information from a single-molecule comprising:
- (a) a molecule having a sequence of elements;
(b) a single-molecule sequencer comprising a polymerase having at least one tag associated therewith;
(c) an excitation source adapted to excite at least one tag on the polymerase or on the monomers; and
(d) a detector adapted to detect a response from the tag on the polymerase;
where the response from at least one tag changes during polymerization of a complementary sequence of elements representing the element sequence of the molecule.
- (a) a molecule having a sequence of elements;
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39. A system for determining sequence information from an individual molecule comprising:
- (a) a molecule having a sequence of elements;
(b) a single-molecule sequencer comprising a polymerase having at least one fluorescent tag associated therewith;
(c) an excitation light source adapted to excite the at least one fluorescent tag on the polymerase or on the monomers; and
(d) a fluorescent light detector adapted to detect at least an intensity of emitted fluorescent light from the at least one fluorescent tag on the polymerase;
where the intensity change of at least one fluorescent tag emits or fails to emit fluorescent light each time a new nucleotide or nucleotide analog is polymerized into a complementary sequence and either the duration of the emission or lack of emission or the wavelength range of the emitted light evidences the particular nucleotide or nucleotide analog polymerized into the sequence so that at the completion of the sequencing the data stream is retrieved.
- (a) a molecule having a sequence of elements;
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40. A single-molecule DNA sequencing system of this invention, a single tag is attached to an appropriate site on a polymerase and a unique tag is attached to each of the four nucleotides:
- dATP, dTTP, dCTP and dGTP. The tags on each dNTPs are designed to have a unique emission signature (i.e., different emission frequency spectrum or color), which is directly detected upon incorporation. As a tagged dNTP is incorporated into a growing DNA polymer, a characteristic fluorescent signal or base emission signature is emitted due to the interaction of polymerase tag and the dNTP tag. The fluorescent signals, i.e., the emission intensity and/or frequency, are then detected and analyzed to determine DNA base sequence.
Specification