Methods and compositions for nucleic acid amplification
View Patent ↗Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products.
1. A method for simultaneously amplifying at least two different target nucleic acid sequences contained in a sample comprising the steps of:
(a) contacting a sample with at least two different target capture oligomers and with at least two different amplification oligomer complexes;
(i) wherein each of said target capture oligomers and each of said amplification oligomer complexes hybridize to different target nucleic acid sequences,
(ii) wherein each of said amplification oligomer complexes comprises a first amplification oligomer member having a first target specific sequence that is directly joined to a second amplification oligomer member having a second target specific sequence;
(b) contacting said at least two target capture oligomers in step (a) with a solid support and immobilized probe for performing a target capture;
(c) washing said sample after target capture;
(d) pre-amplifying said target nucleic acid sequences using said amplification oligomer complexes, under conditions configured to maintain the amplification oligomer complexes, thereby generating first amplification products for each target nucleic acid hybridized by an amplification oligomer complex;
(e) splitting said pre-amplified sample into at least two separate secondary target specific amplification reactions;
(f) amplifying at least two of said pre-amplified samples generated in step (d) using target specific amplification oligomers that are not joined, thereby generating second amplification products.
2. The method of claim 1 , wherein said amplifying reaction at step (f) is a quantitative amplification reaction whereby an amplification product is detected during the amplification reaction.
3. The method of claim 1 , wherein said amplifying reaction at step (f) is an exponential amplification reaction.
4. The method of claim 1 , wherein said pre-amplifying reaction at step (d) is accomplished using a substantially isothermal amplification reaction.
5. The method of claim 1 , wherein said secondary amplification reaction at step (f) is accomplished using a substantially isothermal amplification reaction.
6. The method of claim 1 , wherein said first amplification products generated at step (d) are divided into a number of secondary target specific amplification reactions that equals the number of different amplification oligomer complexes used in step (a).
7. The method of claim 1 , wherein said amplification oligomer complex is a DH-complex.
8. The method of claim 1 , wherein said amplification oligomer complex comprises a first amplification oligomer member that is a non-promoter primer comprising a target specific sequence joined on its 5′ end to a linking member for linking said first amplification oligomer member to a second amplification oligomer member of said amplification oligomer complex.
9. The method of claim 8 , wherein said second amplification oligomer member is a promoter primer oligomer, optionally comprising a blocked 3′ terminus.
10. The method of claim 9 , wherein said first oligomer member comprises a linking member that is a nucleotide sequence that is complementary to a portion of a nucleotide sequence of said second amplification oligomer member.
11. The method of claim 10 , wherein said first oligomer member comprises a linking member that is a nucleotide sequence that is complementary to a promoter sequence of said second amplification oligomer member.
12. The method of claim 1 , wherein said pre-amplification reaction method further comprises using a blocker oligomer.
13. The method of claim 1 , wherein amplifications product from said secondary amplification reaction is detected using a probe based detection step.
14. The method of claim 13 , wherein said probe based detection step is a real-time detection step.
15. The method of claim 1 , wherein each of said amplification oligomer complexes hybridizes to different regions on the same target nucleic acid.
16. The method of claim 1 , wherein each of said amplification oligomer complexes hybridizes to different target nucleic acids.
17. The method of claim 1 , wherein either said first or said second amplification oligomer member of said amplification oligomer complex, comprises a universal tag sequence located 5′ to said target specific sequence and wherein said secondary amplification reaction further includes a universal amplification oligomer targeting said universal tag sequence.
18. The method of claim 1 , wherein both said first and said second amplification oligomer members of said amplification oligomer complex, comprise a universal tag sequence located 5′ to said target specific sequence and wherein said secondary amplification reaction includes a universal amplification oligomer targeting said universal tag sequence.
19. The method of claim 1 , wherein said first or second amplification oligomer member of said amplification oligomer complex, further comprises a promoter provider sequence located 5′ to said target specific sequence.
20. The method of claim 2 , wherein the amplification product is detected during the amplification process using a detection probe, a molecular beacon, a molecular torch or a hybridization switch probe.
21. The method of claim 1 , further comprising a step for detecting amplification product, wherein the amplification product is detection using a detection probe oligomer, using mass spectrometry, using nucleic acid sequencing or using gel electrophoresis.