Methods and compositions for nucleic acid amplification
View Patent ↗Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.
1. A target capture reaction mixture for separating a target nucleic acid from a sample, the reaction mixture comprising:
a. a target specific universal (TSU) primer complex made up of
i. a TSU promoter oligonucleotide comprising a 5′ promoter sequence, an internal first universal sequence (U1), and a 3′ first target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoter primer that has a 3′ terminus that is capable of being extended by a polymerase, or is a TSU promoter provider oligonucleotide that has a blocked 3′ terminus that is incapable of being extended by a polymerase, directly or indirectly joined to,
ii. a TSU non-promoter primer oligonucleotide made up of a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1,
wherein the TSU promoter oligonucleotide is joined to the TSU non-promoter primer via:
(A) a covalent linkage that is a polynucleotide linker sequence or a non-nucleotide a basic linker compound;
(B) a hybridization complex between a first sequence on the TSU promoter oligonucleotide and a second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter oligonucleotide; or
(C) a hybridization complex that includes an S-oligonucleotide that contains a first sequence complementary to a sequence in the TSU promoter oligonucleotide and a second sequence complementary to a sequence in the TSU non-promoter primer oligonucleotide; and
b. a target specific capture oligonucleotide that contains a target specific sequence (TS3) that hybridizes specifically to a sequence in the target nucleic acid that is different from the sequence in the target nucleic acid that hybridizes to the TS sequence of the TSU promoter oligonucleotide or the TS sequence of the TSU non-promoter primer, and contains a means for binding the target nucleic acid to a solid support.
2. The target capture reaction mixture of claim 1 , wherein the TSU promoter oligonucleotide is directly joined to the TSU non-promoter primer oligonucleotide via the hybridization complex between the first sequence on the TSU promoter oligonucleotide and the second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter oligonucleotide.
3. The target capture reaction mixture of claim 1 , wherein the means for binding the target nucleic acid to a solid support is an immobilized probe-binding region that binds to an immobilized probe attached to the solid support, wherein the immobilized probe-binding region binds to the immobilized probe by a specific binding pair interaction.
4. The target capture reaction mixture of claim 3 , wherein the immobilized probe-binding region is a nucleic acid sequence.
5. The target capture reaction mixture of claim 4 , wherein the immobilized probe-binding region comprises a poly-A sequence, wherein said poly-A sequence is attached to the 3′ end of the target specific capture oligonucleotide's target specific sequence.
6. The target capture reaction mixture of claim 1 , wherein the reaction mixture further comprises the solid support.
7. The target capture reaction mixture of claim 5 , wherein the reaction mixture further comprises the solid support, and wherein the solid support comprises an immobilized poly-T sequence.
8. The target capture reaction mixture of claim 1 , further comprising a blocker oligonucleotide that hybridizes specifically to a sequence in a target nucleic acid strand that is different than the sequence that the TS sequence of the TSU promoter oligonucleotide or the TS sequence of the TSU non-promoter primer oligonucleotide binds to in the target nucleic acid strand, wherein the blocker oligonucleotide has a 3′ blocked terminus that is not capable of being extended by a polymerase.
9. The target capture reaction mixture of claim 1 , further comprising at least one of
c. a universal promoter primer made up of a 5′ promoter sequence and a 3′ universal sequence that is the same as the universal sequence of the TSU promoter oligonucleotide, and
d. a universal primer made up of a universal sequence that is the same as the universal sequence of the TSU non-promoter primer oligonucleotide.
10. The target capture reaction mixture of claim 1 , wherein the mixture is for separating multiple different target nucleic acids from a sample, the reaction mixture comprising at least two different target specific capture oligomers and at least two different target specific universal (TSU) primer complexes, each specific for its respective target nucleic acid.
11. A method of amplifying a target nucleic acid comprising the steps of:
a. mixing a target nucleic acid with a target capture reaction mixture comprising a target specific universal (TSU) primer complex and a target specific capture oligonucleotide, wherein the TSU primer complex is made up of
i. a TSU promoter primer oligonucleotide comprising a 5′ promoter sequence, an internal first universal sequence (U1), and a 3′ first target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, and a 3′ terminus that is capable of being extended by a polymerase, directly or indirectly joined to,
ii. a TSU non-promoter primer oligonucleotide made up of a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1,
wherein the TSU promoter primer oligonucleotide is joined to the TSU non-promoter primer via:
(A) a covalent linkage that is a polynucleotide linker sequence or a non-nucleotide a basic linker compound;
(B) a hybridization complex between a first sequence on the TSU promoter oligonucleotide and a second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter oligonucleotide; or
(C) a hybridization complex that includes an S-oligonucleotide that contains a first sequence complementary to a sequence in the TSU promoter oligonucleotide and a second sequence complementary to a sequence in the TSU non-promoter primer oligonucleotide;
and wherein the target specific capture oligonucleotide contains a target specific sequence (TS3) that hybridizes specifically to a sequence in the target nucleic acid that is different from the sequence in the target nucleic acid that hybridizes to the TS sequence of the TSU promoter primer oligonucleotide or the TS sequence of the TSU non-promoter primer, and contains an immobilized probe-binding region that binds to an immobilized probe attached to a solid support, wherein the immobilized probe-binding region binds to the immobilized probe by a specific binding pair interaction;
b. hybridizing each of the TSU promoter primer oligonucleotide and the target specific capture oligonucleotide to the target nucleic acid via the TS1 and TS3 sequences, respectively ;
c. binding the target specific capture oligonucleotides immobilized probe-binding region to the immobilized probe, thereby immobilizing the target nucleic acid with hybridized TSU primer complex to the solid support;
d. separating the support from the mixture, thereby isolating the target nucleic acid with hybridized TSU primer complexes away from unhybridized TSU primer complexes and other sample components;
e. synthetically extending the 3′ terminus of the TS1 by using a polymerase in vitro nucleic acid synthesis in which the target nucleic acid is a template to make a first cDNA strand;
f. hybridizing the TSU non-promoter primer oligonucleotide of the TSU primer complex to the first cDNA strand by specific hybridization of the TS2 to its target sequence contained in the first cDNA strand; and
g. synthetically extending the 3′ terminus of the TS2 sequence hybridized to the first cDNA strand by a polymerase in vitro nucleic acid synthesis to make a second DNA strand, thereby making a substantially double-stranded DNA that contains a functional promoter sequence and the U1 sequence;
h. enzymatically transcribing RNA transcripts from the functional promoter sequence of the substantially double-stranded DNA to make RNA transcripts that contain a 5′ U1 region sequence, a TS1 sequence, a complement of the second target specific sequence, and a 3′ universal sequence that is complementary to the U2 sequence.
12. The method of claim 11 , wherein the TSU promoter primer oligonucleotide is directly joined to the TSU non-promoter primer oligonucleotide via the covalent linkage.
13. The method of claim 11 , wherein the TSU promoter primer oligonucleotide is directly joined to the TSU non-promoter primer oligonucleotide via the hybridization complex between the first sequence on the TSU promoter primer oligonucleotide and the second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter primer oligonucleotide.
14. The method of claim 11 , wherein the immobilized probe-binding region is a nucleic acid sequence.
15. The method of claim 14 , wherein the immobilized probe-binding region comprises a poly-A sequence, wherein said poly-A sequence is attached to the 3′ end of the target specific capture oligonucleotide's target specific sequence, and wherein the solid support comprises an immobilized poly-T sequence.
16. A method of capturing a target nucleic acid for use in a subsequent amplification process, comprising the steps of:
a. mixing a target nucleic acid with a target capture reaction mixture comprising a target specific universal (TSU) primer complex and a target specific capture oligonucleotide, wherein the TSU primer complex is made up of
i. a TSU promoter oligonucleotide comprising a 5′ promoter sequence, an internal first universal sequence (U1), and a 3′ first target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoter primer that has a 3′ terminus that is capable of being extended by a polymerase, or is a TSU promoter provider oligonucleotide that has a blocked 3′ terminus that is incapable of being extended by a polymerase, directly or indirectly joined to,
ii. a TSU non-promoter primer oligonucleotide made up of a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1,
wherein the TSU promoter oligonucleotide is joined to the TSU non-promoter primer via:
(A) a covalent linkage that is a polynucleotide linker sequence or a non-nucleotide a basic linker compound;
(B) a hybridization complex between a first sequence on the TSU promoter oligonucleotide and a second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter oligonucleotide; or
(C) a hybridization complex that includes an S-oligonucleotide that contains a first sequence complementary to a sequence in the TSU promoter oligonucleotide and a second sequence complementary to a sequence in the TSU non-promoter primer oligonucleotide;
and wherein the target specific capture oligonucleotide contains a target specific sequence (TS3) that hybridizes specifically to a sequence in the target nucleic acid that is different from the sequence in the target nucleic acid that hybridizes to the TS sequence of the TSU promoter oligonucleotide or the TS sequence of the TSU non-promoter primer, and contains an immobilized probe-binding region that binds to an immobilized probe attached to a solid support, wherein the immobilized probe-binding region binds to the immobilized probe by a specific binding pair interaction;
b. hybridizing each of the TSU promoter oligonucleotide and the target specific capture oligonucleotide to the target nucleic acid via the TS1 and TS3 sequences, respectively;
c. binding the target specific capture oligonucleotide's immobilized probe-binding region to the immobilized probe, thereby immobilizing the target nucleic acid with hybridized TSU primer complex to the solid support;
d. separating the support from the mixture, thereby isolating the target nucleic acid with hybridized TSU primer complexes away from unhybridized TSU primer complexes and other sample components.
17. The method of claim 16 , wherein the TSU promoter oligonucleotide is directly joined to the TSU non-promoter primer oligonucleotide via the covalent linkage.
18. The method of claim 16 , wherein the TSU promoter oligonucleotide is directly joined to the TSU non-promoter primer oligonucleotide via the hybridization complex between the first sequence on the TSU promoter oligonucleotide and the second sequence on the TSU non-promoter primer that is complementary to the first sequence on the TSU promoter oligonucleotide.
19. The method of claim 16 , wherein the immobilized probe-binding region is a nucleic acid sequence.
20. The method of claim 19 , wherein the immobilized probe-binding region comprises a poly-A sequence, wherein said poly-A sequence is attached to the 3′ end of the target specific capture oligonucleotide's target specific sequence, and wherein the solid support comprises an immobilized poly-T sequence.