US 6,686,156 B2Granted

Methods and compositions for transcription-based nucleic acid amplification

AssigneeNuGEN Technologies, Inc.

InventorNurith Kurn

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Abstract

Methods for isothermal exponential amplification of a target polynucleotide are disclosed. The methods employ two transcription modules, the first module providing linear amplification resulting in RNA transcripts, and a second module providing for further (generally cyclical) amplification resulting in more RNA transcripts. In one aspect, the amplification of the first module is composite primer based. In a second aspect, the amplification of the first module is based on target switching to generate a primer extension product comprising a promoter sequence. In all aspects, the RNA transcripts of the first transcription module are subjected to further amplification by creating an intermediate product comprising a double stranded promoter region from which transcription can occur. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification results.

Claims61

Dependent claims

A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) hybridizing a target polynucleotide comprising the nucleic acid sequence of interest with a first primer, wherein said first primer is a composite primer comprising an RNA portion and a 3′ DNA portion, wherein the target polynucleotide is single-stranded;
(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide (4 sequence to a region of the target polynucleotide 5′ with respect to a site for hybridization of the first primer to the target polynucleotide;
(c) extending the first primer with an enzyme comprising DNA-dependent DNA polymerase activity to generate a complex comprising a first primer extension product and target polynucleotide;
(d) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the target polynucleotide and repeating primer extension by strand displacement to produce a displaced primer extension product;
(e) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising a sequence complementary to the displaced primer extension products is produced;
(f) hybridizing a second primer to the RNA transcript of step (e);
(g) extending the second primer with an enzyme comprising RNA-dependent DNA polymerase activity to generate a complex comprising a second primer extension product and the RNA transcript;
(h) cleaving RNA in the complex of step (g) with an enzyme that cleaves RNA in an RNA/DNA hybrid;
(i) hybridizing the second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising the nucleic acid sequence of interest is produced;
whereby multiple copies of the nucleic acid sequence of interest are produced.

7 more elements

A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) combining:
an RNA transcript, wherein said RNA transcript is produced by a method comprising the steps of:
(i) hybridizing a target polynucleotide comprising the nucleic acid sequence of interest with a first primer, wherein said first primer is a composite primer comprising an RNA portion and a 3′ DNA portion, wherein the target polynucleotide is single-stranded;
(ii) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the target polynucleotide 5′ with respect to a site for hybridization of the first primer to the target polynucleotide;
(iii) extending the first primer with an enzyme comprising DNA-dependent DNA polymerase activity to generate a complex comprising a first primer extension product and target polynucleotide;
(iv) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the target polynucleotide and repeating primer extension by strand displacement to produce a displaced primer extension product;
(v) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising a sequence complementary to the displaced primer extension products is produced;
a second primer that is hybridizable to a sense RNA transcript comprising the nucleic acid sequence of interest;
an enzyme comprising RNA-dependent DNA polymerase activity;
an enzyme that cleaves RNA from an RNA/DNA hybrid;
a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product;
and
an RNA polymerase;
and
(b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.

12 more elements

A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) combining:
a first primer, wherein the first primer is a composite primer that is hybridizable to a target polynucleotide, and wherein the composite primer comprises an RNA portion and a 3′ DNA portion;
optionally a polynucleotide comprising a termination polynucleotide sequence that is hybridizable to a region of the target polynucleotide which is 5′ with respect to hybridization of the composite primer to the target polynucleotide;
an enzyme comprising DNA-dependent DNA polymerase activity;
an enzyme that cleaves RNA from an RNA/DNA hybrid;
a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a first primer extension product;
an RNA polymerase;
a second primer that is hybridizable to a sense RNA transcript comprising the nucleic acid sequence of interest;
an enzyme comprising RNA-dependent DNA polymerase activity;
and
a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product;
and
(b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, displacement of a first primer extension product from a complex comprising a first primer extension product and target polynucleotide when its RNA is cleaved and a composite primer binds to the target polynucleotide in the complex, hybridization of a propromoter polynucleotide to a second primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.

10 more elements

A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target polynucleotide comprising the nucleic acid sequence of interest with an enzyme comprising DNA-dependent DNA polymerase activity to generate a complex comprising a first primer extension product and target polynucleotide, wherein said first primer is a composite primer comprising an RNA portion and a 3′ DNA portion;
(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the target polynucleotide 5′ with respect to a site for hybridization of the first primer to the target polynucleotide;
(c) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the target polynucleotide and repeating primer extension by strand displacement to produce a displaced primer extension product;
(e) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising sequences complementary to the displaced primer extension products is produced;
(f) generating a second primer extension product complementary to the RNA transcript;
(g) hybridizing the second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising the nucleic acid sequence of interest is produced;
whereby multiple copies of the nucleic acid sequence of interest are produced.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application: 09/893,191Filed 26 Jun 2001 · us provisional application
Priority: 26 Jun 2000
Published: US 2002/0058270 A116 May 2002
Granted: 3 Feb 2004
Term ends: Not estimatedThe source records don’t support an estimate.

Classification3 codes

C12Q 1/6865
Nucleic acid amplification reactions: promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence…
C12Q 1/6876
Involving nucleic acids: nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms