Multiphase nucleic acid amplification
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 amplifying a target nucleic acid sequence present in a sample, the method comprising the steps of:
(a) performing a first phase linear amplification reaction that amplifies the target nucleic acid sequence under conditions that do not support exponential amplification thereof,
wherein the first phase linear amplification reaction generates a first amplification product;
(b) performing a second phase amplification reaction under conditions allowing exponential amplification of the first amplification product, thereby generating a second amplification product that comprises the target nucleic acid sequence or the complement thereof; and
(c) detecting, with a sequence-specific hybridization probe at regular time intervals, synthesis of the second amplification product while the second phase amplification reaction is occurring.
2. The method of claim 1 ,
wherein the first phase linear amplification reaction takes place in a first phase amplification reaction mixture,
wherein the second phase amplification reaction takes place in a second phase amplification reaction mixture, and
wherein the first phase amplification reaction mixture comprises an agent that inhibits exponential amplification of the target nucleic acid sequence.
3. The method of claim 1 ,
wherein the first phase linear amplification reaction takes place in a first phase amplification reaction mixture,
wherein the second phase amplification reaction takes place in a second phase amplification reaction mixture, and
wherein the second phase amplification reaction mixture comprises at least one component required for exponential amplification that is substantially absent or depleted from the first phase amplification reaction mixture after performing step (a) and before performing step (b).
4. The method of claim 3 , wherein step (a) comprises performing the first phase linear amplification reaction to amplify the target nucleic acid sequence by 10-fold to 10,000-fold in the first phase amplification reaction mixture before performing step (b).
5. The method of claim 3 , wherein step (c) comprises detecting with a fluorescently labeled hybridization probe.
6. The method of claim 3 , further comprising the step of (d) quantifying the target nucleic acid sequence in the sample.
7. The method of claim 6 , wherein step (d) comprises comparing results obtained in step (c) with a calibration curve.
8. The method of claim 3 , wherein before step (a) there are the steps of:
contacting the sample with a first amplification oligonucleotide, whereby the first amplification oligonucleotide hybridizes to the target nucleic acid sequence to form a pre-amplification hybrid;
capturing the pre-amplification hybrid onto a solid support; and
washing the solid support to remove any of the first amplification oligonucleotide that did not hybridize to the target nucleic acid sequence, thereby isolating the pre-amplification hybrid.
9. The method of claim 8 , wherein step (b) comprises providing the first amplification product with an amplification oligonucleotide that is substantially absent or depleted from the first phase amplification reaction mixture after performing step (a) and before performing step (b).
10. The method of claim 9 , wherein the amplification oligonucleotide provided to the first amplification product is the first amplification oligonucleotide.
11. The method of claim 8 , wherein the first amplification oligonucleotide comprises a tag sequence, said tag sequence comprising any of a 5′ promoter sequence recognized by an RNA polymerase, an amplification primer binding site, a specific binding site for target capture, or a sequencing primer binding site.
12. The method of claim 8 , wherein the first amplification oligonucleotide comprises a tag sequence, said tag sequence comprising a 5′promoter sequence recognized by a T7 RNA polymerase.
13. The method of claim 3 , wherein said at least one component is selected from the group consisting of: an amplification oligonucleotide, a reverse transcriptase, a polymerase, a nuclease, a phosphorylase, an enzyme co-factor, a chelator, ribonucleotide triphosphates, deoxyribonucleotide triphosphates, magnesium ion, and a salt.
14. The method of claim 3 , wherein said at least one component comprises an amplification oligonucleotide.
15. The method of claim 14 , wherein the amplification oligonucleotide comprises a 5′ tag sequence.
16. The method of claim 3 , wherein the first phase amplification reaction mixture comprises an enzyme with RNase H activity.
17. The method of claim 3 , wherein step (b) comprises contacting the first amplification product with an amplification oligonucleotide that is substantially absent or depleted from the first phase amplification reaction mixture after performing step (a) and before performing step (b).
18. The method of claim 1 , wherein the first phase linear amplification reaction and the second phase amplification reaction are isothermal amplification reactions.
19. The method of claim 1 , wherein the first phase linear amplification reaction and the second phase amplification reaction are performed in the same reaction vessel.
20. The method of claim 1 , wherein the first amplification product comprises DNA.
21. The method of claim 1 , wherein the second amplification product comprises RNA.
22. The method of claim 1 , wherein the target nucleic acid sequence is an RNA sequence.
23. The method of claim 1 , wherein before step (a) there are the steps of:
contacting the sample with a first amplification oligonucleotide, whereby the first amplification oligonucleotide hybridizes to the target nucleic acid sequence to form a pre-amplification hybrid;
capturing the pre-amplification hybrid onto a solid support; and
washing the solid support to remove any of the first amplification oligonucleotide that did not hybridize to the target nucleic acid sequence, thereby isolating the pre-amplification hybrid.
24. The method of claim 23 , wherein the first amplification oligonucleotide comprises a tag sequence, said tag sequence comprising any of a 5′ promoter sequence recognized by an RNA polymerase, an amplification primer binding site, a specific binding site for target capture, or a sequencing primer binding site.
25. The method of claim 23 , wherein the first amplification oligonucleotide comprises a tag sequence, said tag sequence comprising a 5′promoter sequence recognized by a T7 RNA polymerase.
26. A method of amplifying a target nucleic acid sequence present in a sample, the method comprising the steps of:
(a) performing a first phase linear amplification reaction that amplifies the target nucleic acid sequence under conditions that do not support exponential amplification thereof,
wherein the first phase linear amplification reaction generates a first amplification product;
(b) performing a second phase amplification reaction under conditions allowing exponential amplification of the first amplification product, thereby generating a second amplification product that comprises the target nucleic acid sequence or the complement thereof; and
c) detecting the second amplification product while the second phase amplification reaction is occurring,
wherein step (c) comprises detecting with a fluorescently labeled hybridization probe.