Method for the direct, exponential amplification and sequencing of DNA molecules and its application
First Claim
1. A method for sequencing at least a portion of a RNA involving converting said RNA into a DNA and simultaneously amplifying said DNA and generating truncated copies of said DNA for sequencing, comprising the steps of (a) subjecting a mixture A to conversion of said RNA to said DNA and, in a single step, to DNA amplification and generation of truncated copies of said DNA by subjecting the mixture A to a thermocycling reaction, the thermocycling reaction comprises heat denaturation, annealing and synthesis, wherein said mixture A comprises said RNA, a first primer which is able to hybridize with a strand of said DNA, a second primer which is able to hybridize with a strand of DNA complementary to the strand with which the first primer is able to hybridize, wherein at least one of the first and second primers is labelled, a reaction buffer, deoxynucleotides or deoxynucleotide derivatives, wherein said deoxynucleotide derivatives are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules in place of one of dATP, dGTP, dTTP or dCTP, at least one dideoxynucleotide or another terminating nucleotide, and a thermostable DNA polymerase having a reduced, compared to wild-type Taq DNA polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, wherein said thermostable DNA polymerase has (1) a Tabor-Richardson mutation, or a functional derivative thereof, and (2) reverse transcriptase activity,
- to simultaneously make full-length and truncated copies of said DNA, wherein the full-length copies have a length equal to that of at least a portion of said DNA spanning the binding sites of the first and second primers;
(b) separating at least the truncated copies of said DNA to obtain a sequence ladder; and
thereafter (c) reading the sequence ladder to obtain the sequence of said at least a portion of said RNA.
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Abstract
A method is described for the direct, exponential amplification and sequencing (“DEXAS”) of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof. In a preferred embodiment of the method of the invention, direct sequencing of RNA can be performed using one polymerase having a Tabor-Richardson mutation, or a functional derivative thereof, and reverse transcriptase activity. In a more preferred embodiment of the method of the invention, direct sequencing of RNA can be performed in one step, in one vessel.
169 Citations
55 Claims
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1. A method for sequencing at least a portion of a RNA involving converting said RNA into a DNA and simultaneously amplifying said DNA and generating truncated copies of said DNA for sequencing, comprising the steps of
(a) subjecting a mixture A to conversion of said RNA to said DNA and, in a single step, to DNA amplification and generation of truncated copies of said DNA by subjecting the mixture A to a thermocycling reaction, the thermocycling reaction comprises heat denaturation, annealing and synthesis, wherein said mixture A comprises said RNA, a first primer which is able to hybridize with a strand of said DNA, a second primer which is able to hybridize with a strand of DNA complementary to the strand with which the first primer is able to hybridize, wherein at least one of the first and second primers is labelled, a reaction buffer, deoxynucleotides or deoxynucleotide derivatives, wherein said deoxynucleotide derivatives are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules in place of one of dATP, dGTP, dTTP or dCTP, at least one dideoxynucleotide or another terminating nucleotide, and a thermostable DNA polymerase having a reduced, compared to wild-type Taq DNA polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, wherein said thermostable DNA polymerase has (1) a Tabor-Richardson mutation, or a functional derivative thereof, and (2) reverse transcriptase activity, - to simultaneously make full-length and truncated copies of said DNA, wherein the full-length copies have a length equal to that of at least a portion of said DNA spanning the binding sites of the first and second primers;
(b) separating at least the truncated copies of said DNA to obtain a sequence ladder; and
thereafter(c) reading the sequence ladder to obtain the sequence of said at least a portion of said RNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 54, 55)
- to simultaneously make full-length and truncated copies of said DNA, wherein the full-length copies have a length equal to that of at least a portion of said DNA spanning the binding sites of the first and second primers;
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32. A kit for sequencing at least a portion of a RNA comprising
deoxynucleotides or deoxynucleotide derivatives, which deoxynucleotide derivatives are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules in place of one of dATP, dGTP, dTTP or dCTP; -
at least one dideoxynucleotide or another terminating nucleotide; and
a thermostable DNA polymerase having a reduced, compared with wild-type Taq polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, wherein said thermostable DNA polymerase has (1) a Tabor-Richardson mutation, or a functional derivative thereof, and (2) reverse transcriptase activity. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53)
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Specification