Methods and compositions for nucleic acid amplification
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 non-promoter primer oligonucleotide having a first 3′ target specific sequence and a promoter oligonucleotide having a second 3′ target specific sequence and a 5′ promoter sequence, wherein the promoter oligonucleotide optionally comprises a blocked 3′ terminus, and wherein the non-promoter primer oligonucleotide is directly or indirectly joined to the promoter oligonucleotide;
(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 the non-promoter primer oligonucleotide is joined to the promoter oligonucleotide via:
a direct hybridization (DH) complex between a first sequence in the non-promoter primer oligonucleotide and a second sequence in the promoter oligonucleotide that is complementary to the first sequence in the non-promoter primer oligonucleotide;
a hybridization complex comprising an S-oligonucleotide, wherein the S-oligonucleotide contains a first sequence complementary to a sequence in the promoter oligonucleotide and a second sequence complementary to a sequence in the non-promoter primer oligonucleotide; or
a non-nucleotide linker.
3. The method of claim 2 , wherein the non-promoter primer oligonucleotide is joined to the promoter oligonucleotide via the DH complex.
4. The method of claim 3 , wherein the first and second sequences of the DH complex are a 5′ sequence in the non-promoter primer oligonucleotide that is complementary to all or a portion of the promoter sequence in the promoter oligonucleotide.
5. The method of claim 1 , wherein the amplifying reaction at step (f) is a quantitative amplification reaction whereby an amplification product is detected during the amplification reaction.
6. The method of claim 1 , wherein the amplifying reaction at step (f) is an exponential amplification reaction.
7. The method of claim 1 , wherein the amplification reaction at step (f) is accomplished using a substantially isothermal amplification reaction.
8. The method of claim 1 , wherein a first amplification product generated in step (b) for a target nucleic acid hybridized by an amplification oligomer complex is from 100 to 10000 RNA transcripts.
9. The method of claim 1 , where the 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).
10. The method of claim 1 , wherein the amplification oligomer complexes are formed before being added into the pre-amplifying reaction at step (d).
11. The method of claim 1 , wherein the amplification oligomer complexes are formed and then added into a target capture reaction mixture at step (a).
12. The method of claim 1 , wherein the amplification oligomer complexes are formed under hybridizing conditions during step (a).
13. The method of claim 1 , wherein the pre-amplifying reaction at step (d) further comprises using a blocker oligomer.
14. The method of claim 1 , further comprising detecting the second amplification products generated at step (f).
15. The method of claim 14 , wherein the second amplification products are detected using a probe-based detection step.
16. The method of claim 15 , wherein the probe-based detection step is a real-time detection step.
17. The method of claim 16 , wherein the second amplification products are detected using a detection probe oligomer selected from the group consisting of a molecular beacon, a molecular torch, and a hybridization switch probe.
18. The method of claim 14 , wherein the second amplification products are detected using a detection probe oligomer, mass spectrometry, nucleic acid sequencing, or gel electrophoresis.
19. The method of claim 1 , wherein each of the amplification oligomer complexes hybridizes to a different region on the same target nucleic acid.
20. The method of claim 1 , wherein each of the amplification oligomer complexes hybridizes to a different target nucleic acid.