Enzymatic nucleic acid synthesis: compositions and methods
Nucleotide triphosphate probes containing a molecular and/or atomic tag on a a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.
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7 . A method comprising the step of adding a phosphatase to a polymerization reaction to degrade untagged.
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21 . A method of adding a phosphatase to a polymerization reaction to degrade natural nucleotides and where tagged pyrophosphate released upon tagged nucleotide incorporation inhibits pyrophosphorolysis.
22 . A method of adding a phosphatase to a polymerization reaction to degrade non-phosphate labeled nucleotides and where tagged pyrophosphate released upon tagged nucleotide incorporation inhibits pyrophosphorolysis.
23 . A method of adding a phosphatase to a nucleotide mixture including natural nucleotides and phosphate-tagged monomers to degrade untagged nucleotides.
24 . A method of adding a phosphatase to a nucleotide mixture including natural nucleotides and phosphate-tagged monomers to degrade and where tagged pyrophosphate released upon tagged nucleotide incorporation inhibits pyrophosphorolysis.
25 . A method of identifying a base at a target position in a sample DNA sequence comprising the steps of:
providing an extension primer, which hybridizes to the sample DNA either immediately adjacent to or within about 10 bases of the target position and the sample DNA to form a primer-sample DNA extendable duplex,
subjecting the extendable duplex to a polymerase reaction in the presence of a tagged deoxynucleotide or dideoxynucleotide in the presence of a nucleotide-degrading enzyme, where the tagged deoxynucleotide or dideoxynucleotide an atomic and/or molecular tag or moiety having a detectable property attached to and/or associated with a β and/or γ-phosphate and/or a base moiety of the deoxynucleotide or dideoxynucleotide, where the tagged deoxynucleotide or dideoxynucleotide only becomes incorporated and release tagged pyrophosphate (PPi) if it is complementary to the base in the target position,
detecting released of tagged PPi to identify the deoxynucleotide or dideoxynucleotide being incorporated.
26 . A method of single molecule detection comprising the steps of:
tethering an unlabeled, single-stranded target nucleic acid with a primer hybridized thereto to a surface of a solid support,
flowing an aqueous solution comprising a polymerase or reverse transcriptase and tagged dNTP types across the surface, where the tag on the dNTP has a detectable property,
detecting the detectable property of the tag during one or a series of dNTP incorporations, and
relating the detected dNTP or dNTPs to one or a sequence of nucleotides in the nucleic acid.
27 . A method of single molecule detection comprising the steps of:
immobilizing an individual polymerase molecule on a support and
bathing the polymerase in a flowing solution comprising: 1) unlabeled, single-stranded DNA fragments hybridized to an oligonucleotide primer and 2) a mixture of tagged deoxynucleotide triphosphate types, where the tag on the dNTP has a detectable property,
detecting the detectable property of the tag during one or a series of dNTP incorporations, and
relating the detected dNTP or dNTPs to one or a sequence of nucleotides in the nucleic acid.
28 . A method of single molecule detection comprising the steps of:
immobilizing a library of oligonucleotides on a solid support, and
bathing the support in a solution comprising: 1) a polymerizing agent, 2) unlabeled, single-stranded DNA fragments hybridized to an oligonucleotide primer and 3) a mixture of tagged deoxynucleotide triphosphate types, where the tag on the dNTP has a detectable property,
detecting the detectable property of the tag during one or a series of dNTP incorporations, and
relating the detected dNTP or dNTPs to one or a sequence of nucleotides in the nucleic acid.
29 . A method of single molecule detection comprising the steps of:
immobilizing an individual polymerase molecule on a support and
bathing the polymerase in a solution comprising: 1) nicked double strained DNA, where the nicking is either affected via chemical means selected from the group consisting of Fe-EDTA and DNases, and 2) a mixture of tagged deoxynucleotide triphosphate types, where the tag on the dNTP has a detectable property,
detecting the detectable property of the tag during one or a series of dNTP incorporations, and
relating the detected dNTP or dNTPs to one or a sequence of nucleotides in the nucleic acid.