Methods And Compositions For Transcription-Based Nucleic Acid Amplification

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.

Description

CROSS-REFERENCE[0001]This application is a divisional of U.S. Ser. No. 10 / 686,466, filed Oct. 14, 2003, which is a divisional of U.S., Ser. No. 09 / 893,191, filed Jun. 26, 2001, now U.S. Pat. No. 6,686,156, which claims the priority benefit of provisional patent applications 60 / 277,748, filed Mar. 21, 2001 and 60 / 213,908 filed Jun. 26, 2000, all of which are incorporated herein by reference in their entirety.TECHNICAL FIELD[0002]This invention relates to the filed of polynucleotide amplification. More particularly, the invention provides methods, compositions and kits for amplifying (i.e., making multiple copies) target polynucleotide sequences which involve transcription, and employ either an RNA / DNA composite primer and / or a target switch polynucleotide.BACKGROUND ART[0003]The development of methods for nucleic acid amplification and detection of amplification products have advanced the detection, identification, quantification and sequence analyses of nucleic acids in recent years...

AI Technical Summary

Benefits of technology

[0102]Amplification methods of the invention have the additional advantage of being resistant to non-specific incorporation of a promoter sequence into primer extension products which can lead to non-specific amplification. In the composite primer-based methods, the promoter sequence is introduced by a TSO and / or another propromoter polynucleotide as described herein. In the propromoter TSO-based methods, the promoter sequence is introduced by a propromoter TSO in the first transcription module, and another propromoter polynucleotide which, as described herein, can be a TSO, in the second transcription module. In the first transcription module of the propromoter TSO-based methods, the formation of a double stranded functional promoter is dependent on primer extension and template switch. The formation of a functional promoter in the second module requires hybridization of the second primer extension product to a propromoter polynucleotide such as a propromoter TSO. The step of propromoter polynucleotide hybridization in the methods of the invention increases the specificity of the process. A non-specific extension product would not be likely to have a sequence (e.g., a 3′ end sequence) which is complementary to a propromoter polynucleotide designed to be hybridizable to a particular sequence in a primer extension product.

Problems solved by technology

Although detection of the presence of a defined nucleic acid sequence, and its sequence analysis, can be carried out by direct probe hybridization to target nucleic acid sequences, the method generally lacks sensitivity when amounts of the target nucleic acid sequence present in the test sample are low.

However, previously known isothermal target amplification methods have severe drawbacks.

Amplification according to the strand displacement amplification (SDA) process requires the presence of sites for defined restriction enzymes, thus limiting its applicability.

Existing transcription-based amplification methods, such as the NASBA and TMA, on the other hand, are limited by the need for incorporation of the RNA polymerase promoter sequence into the amplification product by means of a primer, a process prone to causing non-specific amplification.

Moreover, the mechanism of amplification of a DNA target by these transcription-based amplification methods is not Well established.

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