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 pre-amplification reaction mixture for use in simultaneously amplifying at least two different target nucleic acid sequences contained in a sample to generate first amplification products, wherein the pre-amplification reaction mixture comprises
(a) an enzyme selected from the group consisting of: a polymerase, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, a reverse transcriptase, an RNase, or a combination thereof;
(b) an immobilized probe attached to a solid support;
(c) at least two different target capture oligomers, wherein each of said target capture oligomers comprises (i) a target specific sequence and (ii) an immobilized probe-binding region that binds to the immobilized probe; and
(d) at least two different amplification oligomer complexes, 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 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, and
wherein the pre-amplification reaction mixture does not contain any target-specific amplification oligomers that are not joined to form one of the at least two amplification oligomer complexes.
2. The pre-amplification reaction mixture of claim 1 , wherein at least one of the amplification oligomer complexes is a DH-complex.
3. The pre-amplification reaction mixture of claim 1 , wherein the first amplification oligomer member of at least one of the amplification oligomer complexes 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.
4. The pre-amplification reaction mixture of claim 3 , wherein the second amplification oligomer member comprises a blocked 3′ terminus.
5. The pre-amplification reaction mixture of claim 4 , wherein the linking member is a nucleotide sequence that is complementary to a portion of a nucleotide sequence of the second amplification oligomer member.
6. The pre-amplification reaction mixture of claim 5 , wherein the linking member is a nucleotide sequence that is complementary to a promoter sequence of the second amplification oligomer member.
7. The pre-amplification reaction mixture of claim 1 , wherein said solid support is a magbead.
8. The pre-amplification reaction mixture of claim 1 , further comprising an imidazolium compound in an amount sufficient to provide 0.05M to 4.2M when combined with said sample.
9. The pre-amplification reaction mixture of claim 7 , wherein at least one of the amplification oligomer complexes is a DH-complex.
10. The pre-amplification reaction mixture of claim 7 , wherein the first amplification oligomer member of at least one of the amplification oligomer complexes 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.
11. The pre-amplification reaction mixture of claim 10 , wherein the second amplification oligomer member comprises a blocked 3′ terminus.
12. The pre-amplification 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 pre-amplification 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 pre-amplification reaction mixture of claim 7 , further comprising an imidazolium compound in an amount sufficient to provide 0.05M to 4.2M when combined with said sample.
15. The pre-amplification reaction mixture of claim 8 , wherein at least one of the amplification oligomer complexes is a DH-complex.
16. The pre-amplification reaction mixture of claim 8 , wherein the first amplification oligomer member of at least one of the amplification oligomer complexes 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.
17. The pre-amplification reaction mixture of claim 16 , wherein the second amplification oligomer member comprises a blocked 3′ terminus.
18. The pre-amplification reaction mixture of claim 17 , wherein the linking member is a nucleotide sequence that is complementary to a portion of a nucleotide sequence of the second amplification oligomer member.
19. The pre-amplification reaction mixture of claim 18 , wherein the linking member is a nucleotide sequence that is complementary to a promoter sequence of the second amplification oligomer member.