US 6,605,428 B2Granted

Method for the direct, exponential amplification and sequencing of DNA molecules and its application

AssigneeRoche Diagnostics GmbH

InventorsChristian Kilger, Svante Paabo

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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.

Claims87

Dependent claims

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.

6 more elements

A method for sequencing at least a portion of a DNA involving simultaneously amplifying said DNA and generating truncated copies of said DNA for sequencing, comprising the steps of

(a) subjecting a mixture, in a single step, to DNA amplification and generation of truncated copies of said DNA by subjecting the mixture to a thermocycling reaction, the thermocycling reaction comprises heat denaturation, annealing and synthesis, wherein said mixture comprises said DNA,
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 molecule, in place of one of dATP, dGTP, dTTP or dCTP, m
at least one dideoxynucleotide or another terminating nucleotide
a thermostable DNA polymerase having a reduced, compared to wild-type Taq DNA polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, and
at least one polymerase-inhibiting agent against said thermostable DNA polymerase, which polymerase-inhibiting agent loses inhibitory ability, thereby allowing said thermostable DNA polymerase to be active, at a temperature which is at least the temperature at which unspecifically hydridized primers separate from a DNA molecule, 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 DNA.

8 more elements

Dependencies are inferred from the claim text.

Facts

Application: 09/339,104Filed 24 Jun 1999 · continuation in part
Priority: DE 196 53 43920 Dec 1996
Published: US 2003/0134276 A117 Jul 2003
Granted: 12 Aug 2003
Term ends: Not estimatedThe source records don’t support an estimate.

Classification2 codes

C12Q 1/6869
Involving nucleic acids: methods for sequencing
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms