Multiphase nucleic acid amplification
View Patent ↗Improved methods for use in nucleic acid amplification, including multiplex amplification, where the amplification is carried out in two or more distinct phases are disclosed. The first phase amplification reaction preferably lacks one or more components required for exponential amplification. The lacking component is subsequently provided in a second, third or further phase(s) of amplification, resulting in a rapid exponential amplification reaction. The multiphase protocol results in faster and more sensitive detection and lower variability at low analyte concentrations. Compositions for carrying out the claimed methods are also disclosed.
1. A method of quantifying a target nucleic acid sequence in a sample, comprising the steps of:
(a) contacting the sample with a first amplification oligonucleotide, specific for a first portion of the target nucleic acid sequence, under conditions allowing hybridization of the first amplification oligonucleotide to the first portion of the target nucleic acid sequence, thereby generating a pre-amplification hybrid that comprises the first amplification oligonucleotide and the target nucleic acid sequence;
(b) isolating the pre-amplification hybrid by target capture onto a solid support followed by washing to remove any of the first amplification oligonucleotide that did not hybridize to the first portion of the target nucleic acid sequence in step (a);
(c) amplifying, in a first phase amplification reaction mixture, at least a portion of the target nucleic acid sequence of the pre-amplification hybrid isolated in step (b) in a first phase, substantially isothermal, transcription-associated amplification reaction under conditions that support linear amplification thereof, but do not support exponential amplification thereof, thereby resulting in a reaction mixture comprising a first amplification product,
wherein the first phase amplification reaction mixture comprises a second amplification oligonucleotide, the second amplification oligonucleotide being complementary to a portion of an extension product of the first amplification oligonucleotide, and
wherein the first amplification product is not a template for nucleic acid synthesis during the first phase, substantially isothermal, transcription-associated amplification reaction;
(d) combining the reaction mixture comprising the first amplification product with at least one component that participates in exponential amplification of the first amplification product, but that is lacking from the reaction mixture comprising the first amplification product, to produce a second phase amplification reaction mixture,
wherein the second phase amplification reaction mixture additionally comprises a sequence-specific hybridization probe;
(e) performing, in a second phase, substantially isothermal, transcription-associated amplification reaction in the second phase amplification reaction mixture, an exponential amplification of the first amplification product, thereby synthesizing a second amplification product;
(f) detecting, with the sequence-specific hybridization probe at regular time intervals, synthesis of the second amplification product in the second phase amplification reaction mixture; and
(g) quantifying the target nucleic acid sequence in the sample using results from step (f).
2. The method of claim 1 , wherein the first amplification oligonucleotide comprises a 3′ target specific sequence and a 5′ promoter sequence for an RNA polymerase.
3. The method of claim 2 , wherein the RNA polymerase is T7 RNA polymerase.
4. The method of claim 1 , wherein the second amplification oligonucleotide is enzymatically extended in the first phase isothermal transcription-associated amplification reaction.
5. The method of claim 1 , wherein the solid support comprises an immobilized capture probe.
6. The method of claim 1 ,
wherein step (a) further comprises contacting the sample with a target capture oligonucleotide that hybridizes to the target nucleic acid sequence, and
wherein the pre-amplification hybrid comprises the target nucleic acid sequence hybridized to each of the target capture oligonucleotide and the first amplification oligonucleotide.
7. The method of claim 1 , wherein the solid support comprises magnetically attractable particles.
8. The method of claim 1 ,
wherein each of the first and second phase isothermal transcription-associated amplification reactions comprise an RNA polymerase and a reverse transcriptase.
9. The method of claim 1 , wherein the at least one component comprises the first amplification oligonucleotide.
10. The method of claim 1 ,
wherein the first amplification product of step (c) is a cDNA molecule with the same polarity as the target nucleic acid sequence in the sample, and
wherein the second amplification product of step (e) is an RNA molecule.
11. The method of claim 1 , wherein the sequence-specific hybridization probe in step (d) is a conformation-sensitive probe that produces a detectable signal when hybridized to the second amplification product.
12. The method of claim 1 , wherein the sequence-specific hybridization probe in step (d) is a fluorescently labeled sequence-specific hybridization probe.
13. The method of claim 1 , wherein step (g) comprises quantifying the target nucleic acid sequence in the sample using a linear calibration curve and results from step (f).
14. The method of claim 1 , wherein step (c) comprises amplifying by 10-fold to 10,000-fold, in the first phase amplification reaction mixture.