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 target capture reaction mixture, wherein the reaction mixture comprises at least one target nucleic acid, at least one target capture oligonucleotide and at least one amplification oligomer complex, wherein each of said amplification oligomer complexes comprises a first amplification oligomer member having a first target specific sequence that is joined to a second amplification oligomer member having a second target specific sequence, wherein the first amplification oligomer member is a non-promoter primer and the second amplification oligomer member is a promoter primer, and wherein each of said target capture oligomers and each of said amplification oligomer complexes specifically hybridizes to a different target nucleic acid sequence.
2. The target capture reaction mixture of claim 1 , further comprising at least one solid support.
3. The target capture reaction mixture of claim 2 , wherein said solid support is a magbead.
4. The target capture reaction mixture of claim 1 , wherein said at least one target capture oligomer is a wobble probe.
5. The target capture reaction mixture of claim 1 , further comprising from about 0.05M to about 4.2M of an imidazolium compound.
6. The target capture reaction mixture of claim 1 , wherein at least one of the amplification oligomer complexes is a DH-complex.
7. The target capture reaction mixture of claim 6 , wherein the first amplification oligomer member of at least one of the amplification oligomer complexes is joined on its 5′ end to a linking member for linking the first amplification oligomer member to the second amplification oligomer member of the amplification oligomer complex.
8. The target capture reaction mixture of claim 7 , wherein the linking member is a nucleotide sequence that is complementary to a portion of a nucleotide sequence of the second amplification oligomer member.
9. The target capture reaction mixture of claim 8 , wherein the linking member is a nucleotide sequence that is complementary to a promoter sequence of the second amplification oligomer member.
10. The target capture reaction mixture of claim 1 , wherein the second amplification oligomer member comprises a blocked 3′ terminus.
11. The target capture reaction mixture of claim 1 , wherein the first amplification oligomer member of at least one of the amplification oligomer complexes is joined on its 5′ end to a linking member for linking the first amplification oligomer member to the second amplification oligomer member of the amplification oligomer complex.
12. The target capture reaction mixture of claim 11 , wherein the linking member is a nucleotide sequence that is complementary to a portion of a nucleotide sequence of the second amplification oligomer member.
13. The target capture reaction mixture of claim 12 , wherein the linking member is a nucleotide sequence that is complementary to a promoter sequence of the second amplification oligomer member.
14. The target capture reaction mixture of claim 11 , further comprising from about 0.05M to about 4.2M of an imidazolium compound.
15. A target capture reaction mixture, wherein the reaction mixture comprises
(a) at least one target nucleic acid,
(b) at least one target capture oligonucleotide,
(c) at least one solid support,
(d) at least one amplification oligomer complex, wherein each of said amplification oligomer complexes comprises a first amplification oligomer member having a first target specific sequence that is joined to a second amplification oligomer member having a second target specific sequence, wherein the first amplification oligomer member is a non-promoter primer and the second amplification oligomer member is a promoter primer, wherein the first amplification oligomer member is joined on its 5′ end to a linking member for linking the first amplification oligomer member to the second amplification oligomer member,
wherein each of said target capture oligomers and each of said amplification oligomer complexes specifically hybridizes to a different target nucleic acid sequence, and wherein each target nucleic acid sequence is specifically targeted by one of said target capture oligomers and one of said amplification oligomer complexes.
16. The target capture reaction mixture of claim 15 , wherein the linking member is a nucleotide sequence that is complementary to a portion of a nucleotide sequence of the second amplification oligomer member.
17. The pre-amplification reaction mixture of claim 16 , wherein the linking member is a nucleotide sequence that is complementary to a promoter sequence of the second amplification oligomer member.
18. A target capture reaction mixture, wherein the reaction mixture comprises
(a) at least one target nucleic acid,
(b) at least one target capture oligonucleotide,
(c) at least one solid support, wherein said solid support is a magbead,
(d) at least one amplification oligomer complex, wherein each of said amplification oligomer complexes comprises a first amplification oligomer member having a first target specific sequence that is joined to a second amplification oligomer member having a second target specific sequence, wherein the first amplification oligomer member is a non-promoter primer and the second amplification oligomer member is a promoter primer, wherein the first amplification oligomer member is joined on its 5′ end to a linking member that is a nucleotide sequence that is substantially complementary to the promoter sequence of the second amplification oligomer for linking the first amplification oligomer member to the second amplification oligomer member,
wherein each of said target capture oligomers and each of said amplification oligomer complexes specifically hybridizes to a different target nucleic acid sequence, and wherein each target nucleic acid sequence is specifically targeted by one of said target capture oligomers and one of said amplification oligomer complexes.
19. The target capture reaction mixture of claim 18 , wherein the second amplification oligomer member comprises a blocked 3′ terminus.
20. The target capture reaction mixture of claim 18 , further comprising from about 0.05M to about 4.2M of an imidazolium compound.