Sequence determination by direct detection
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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31 . A method for sequencing a molecular sequence comprising:
supplying a sequence of nucleotides or nucleotide analogs to a sequencer comprising a polymerase and monomer types for the polymerase, each monomer type having a unique fluorescent tag covalently bonded a portion of the monomer that is released after incorporation of the monomer by the polymerase;
exciting the fluorescent tag with light from an excitation light source;
detecting a property of emitted fluorescent light from a tag before, during and/or after monomer incorporation via a fluorescent light detector; and
converting the detected property into an identity of the incorporated monomer complementary to a nucleotide in the sequence.
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35 . The method of claim 31 , wherein the detectable property is a duration of the emitted fluorescent light, an intensity of the emitted fluorescent light and/or a frequency spectrum of the emitted fluorescent light.
36 . The method of claim 35 , wherein the detectable property changes when the monomer is incorporated.
37 . The method of claim 36 , wherein the change in detectable property occurs after monomer incorporation.
38 . The method of claim 37 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β, γ and/or terminal phosphate thereof and where the detectable property is derived from the released portion.
39 . The method of claim 37 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β phosphate thereof.
40 . The method of claim 37 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the γ phosphate thereof.
41 . The method of claim 37 , wherein the monomers nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the terminal phosphate thereof.
42 . A method for sequencing a molecular sequence comprising:
supplying a sequence of nucleotides or nucleotide analogs to a sequencer comprising a polymerase and monomer types for the polymerase, each monomer type having a unique fluorescent tag covalently bonded to a portion of the monomer that is released after incorporation of the monomer by the polymerase;
exciting the fluorescent tag with light from an excitation light source;
detecting a property of emitted fluorescent light before, during and/or after a series of monomer incorporations via a fluorescent light detector; and
converting the detected properties into identities of the series of incorporated monomers complementary to a corresponding nucleotide series in the sequence.
43 . The method of claim 42 , wherein the detectable property is a duration of the emitted fluorescent light, an intensity of the emitted fluorescent light and/or a frequency spectrum of the emitted fluorescent light.
44 . The method of claim 43 , wherein the detectable property changes when the monomer is incorporated.
45 . The method of claim 44 , wherein the change in detectable property occurs after monomer incorporation.
46 . The method of claim 45 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β, γ and/or terminal phosphate thereof and where the detectable property is derived from the released portion.
47 . The method of claim 45 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the β phosphate thereof.
48 . The method of claim 45 , wherein the monomers are nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the γ phosphate thereof.
49 . The method of claim 45 , wherein the monomers nucleotides or nucleotide analogs and the unique fluorescent tags are covalently bonded to the terminal phosphate thereof.