Methods and compositions for nucleic acid amplification

A method of amplifying a target nucleic acid comprising the steps of:

• a. mixing a target nucleic acid with a target specific universal (TSU) primer complex 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, joined to
• ii. a TSU non-promoter primer oligonucleotide comprising a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1,
• iii. wherein the TSU promoter primer oligonucleotide is joined to the TSU non-promoter primer oligonucleotide:
• (A) via a covalent linkage that is a polynucleotide linker sequence, a non-nucleotide abasic linker compound or a linker containing both nucleotide and non-nucleotide residues, or
• (B) via 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) via 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,
• b. hybridizing the TSU promoter primer oligonucleotide of the TSU primer complex to a target sequence in the target nucleic acid via the TS1 sequence in the TSU promoter primer,
• c. isolating the target nucleic acid with hybridized TSU complex away from unhybridized TSU primer complexes and other sample components,
• d. 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,
• e. 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,
• f. synthetically extending the 3′ terminus of the TS2 sequence hybridized to the first cDNA strand by a polymerase in vitro nucleic acid synthesis to made a second DNA strand, thereby making a substantially double-stranded DNA that contains a functional promoter sequence and the U1 sequence,
• g. 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 (TS2′), and a 3′ universal sequence that is complementary to the U2 sequence (U2′),
• h. hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcript at the U2′ sequence,
• i. synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand,
• j. hybridizing a universal promoter primer oligonucleotide (UP1) that contains a universal sequence U1 to the cDNA made in the previous step at the U1′ sequence,
• k. synthetically extending at least the 3′ terminus of the DNA strand made in step i, by enzymatic in vitro nucleic acid synthesis to make a functional promoter, and
• l. transcribing multiple RNA transcripts from the functional promoter, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis.

A method of amplifying a target nucleic acid comprising the steps of:

• a. mixing a target nucleic acid with a target specific universal (TSU) 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 its target sequence, wherein the TSU promoter oligonucleotide is a TSU promoter provider oligonucleotide that has a blocked 3′ terminus that is incapable of being extended by a polymerase, joined to
• ii. a TSU non-promoter primer oligonucleotide comprising a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1,
• iii. wherein the TSU promoter primer oligonucleotide is joined to the TSU non-promoter primer oligonucleotide:
• (A) via a covalent linkage that is a polynucleotide linker sequence, a non-nucleotide abasic linker compound or a linker containing both nucleotide and non-nucleotide residues, or
• (B) via 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) via 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,
• b. hybridizing the TSU non-promoter primer oligonucleotide of the TSU complex to a target sequence in the target nucleic acid via the TS2 sequence in the TSU non-promoter primer,
• c. optionally hybridizing a blocker oligonucleotide to a sequence on the target nucleic acid, wherein said blocker comprises a blocked 3′ terminus to prevent extension of the blocker by a polymerase and wherein the blocker oligomer is hybridized to the target nucleic acid at a position to terminate polymerase extension of the TSU non-promoter primer,
• d. isolating the target nucleic acid with hybridized TSU complex away from unhybridized TSU primer complexes and other sample components,
• e. synthetically extending the 3′ terminus of the TSU non-promoter primer hybridized to the target nucleic acid 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 promoter provider oligonucleotide of the TSU complex to the first cDNA strand by specific hybridization of the TS1 sequence in the TSU promoter provider oligonucleotide to a target sequence contained in the first cDNA strand,
• g. synthetically extending the 3′ terminus of the first cDNA by using the TSU promoter provider as a template to make 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 to make RNA transcripts that contain a 5′ U1 region sequence, a TS1 sequence, a complement of the second target specific sequence (TS2′), and a 3′ universal sequence that is complementary to the U2 sequence (U2′),
• i. hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcript at the U2′ sequence,
• j. synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to make a cDNA strand,
• k. hybridizing to the U1′ sequence a universal promoter oligonucleotide (UP1) that contains a promoter sequence, a universal sequence U1, and a 3′ blocked end to the cDNA made in step j,
• l. synthetically extending the 3′ terminus of the DNA strand made in step j using the UP1 oligonucleotide as a template to make a functional double-stranded promoter using enzymatic in vitro nucleic acid synthesis, and
• m. transcribing multiple RNA transcripts from the functional promoter, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis.

A method of amplifying two or more target nucleic acids in a sample comprising the steps of:

• a. mixing a sample suspected of containing two or more different target nucleic acids with at least two different a target specific universal (TSU) primer complexes, each TSU primer complex being 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 one of the at least two target nucleic acids, and a 3′ terminus that is capable of being extended by a polymerase, joined to
• ii. a TSU non-promoter primer oligonucleotide comprising a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1 and which binds specifically to the amplification product generated by the TSU promoter primer oligonucleotide to which it is joined,
• iii. wherein the TSU promoter primer oligonucleotide is joined to the TSU non-promoter primer oligonucleotide:
• (A) via a covalent linkage that is a polynucleotide linker sequence, a non-nucleotide abasic linker compound or a linker containing both nucleotide and non-nucleotide residues, or
• (B) via 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) via 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,
• b. hybridizing the various TSU promoter primer oligonucleotides of the TSU primer complexes to target sequences in the target nucleic acids via the TS1 sequence in the TSU promoter primers,
• c. isolating the target nucleic acids with hybridized TSU complexes away from unhybridized TSU primer complexes and other sample components,
• d. 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,
• e. hybridizing the TSU non-promoter primer oligonucleotide of each of the TSU primer complexes to the respective first cDNA strand by specific hybridization of the TS2 to its target sequence contained in the first cDNA strand,
• f. synthetically extending the 3′ terminus of the TS2 sequence hybridized to the first cDNA strand by a polymerase in vitro nucleic acid synthesis to made a second DNA strand, thereby making from each a substantially double-stranded DNA that contains a functional promoter sequence and the U1 sequence,
• g. 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 (TS2′), and a 3′ universal sequence that is complementary to the U2 sequence (U2′),
• h. hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcripts at the U2′ sequence,
• i. synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand,
• j. hybridizing a universal promoter primer oligonucleotide (UP1) that contains a universal sequence U1 to the cDNA made in the previous step at the U1′ sequence,
• k. synthetically extending at least the 3′ terminus of the DNA strand made in step i, by enzymatic in vitro nucleic acid synthesis to make a functional promoter, and
• l. transcribing multiple RNA transcripts from the functional promoter, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis.

A method of amplifying two or more target nucleic acids in a sample comprising the steps of:

• a. mixing a sample suspected of containing two or more different target nucleic acids with at least two target specific universal (TSU) complexes, each TSU complex being 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 one of the at least two target nucleic acids, wherein the TSU promoter oligonucleotide is a TSU promoter provider oligonucleotide that has a blocked 3′ terminus that is incapable of being extended by a polymerase, joined to
• ii. a TSU non-promoter primer oligonucleotide comprising a 5′ second universal sequence (U2) and a 3′ second target specific sequence (TS2) which is different from the TS1 and which binds specifically to the amplification product generated by the TSU promoter oligonucleotide to which it is joined,
• iii. wherein the TSU promoter primer oligonucleotide is joined to the TSU non-promoter primer oligonucleotide:
• (A) via a covalent linkage that is a polynucleotide linker sequence, a non-nucleotide abasic linker compound or a linker containing both nucleotide and non-nucleotide residues, or
• (B) via 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) via 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,
• b. hybridizing the various TSU non-promoter primer oligonucleotides of the TSU complexes to target sequences in the target nucleic acids via the TS2 sequence in the TSU non-promoter primers,
• c. optionally hybridizing a blocker oligonucleotide to a sequence on the target nucleic acids, wherein said blocker comprises a blocked 3′ terminus to prevent extension of the blocker by a polymerase and wherein the blocker oligomer is hybridized to the target nucleic acid at a position to terminate polymerase extension of the TSU non-promoter primer,
• d. isolating the target nucleic acids with hybridized TSU complexes away from unhybridized TSU primer complexes and other sample components,
• e. synthetically extending the 3′ terminus of the TSU non-promoter primers hybridized to the target nucleic acid 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 promoter provider oligonucleotide of the TSU complex to the respective first cDNA strand by specific hybridization of the TS1 sequence in the TSU promoter provider oligonucleotide to a target sequence contained in the first cDNA strand,
• g. synthetically extending the 3′ terminus of the first cDNA by using the TSU promoter provider as a template to make 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 to make RNA transcripts that contain a 5′ U1 region sequence, a TS1 sequence, a complement of the second target specific sequence (TS2′), and a 3′ universal sequence that is complementary to the U2 sequence (U2′),
• i. hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcript at the U2′ sequence,
• j. synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to make a cDNA strand,
• k. hybridizing to the U1′ sequence a universal promoter oligonucleotide (UP1) that contains a promoter sequence, a universal sequence U1, and a 3′ blocked end to the cDNA made in step j,
• l. synthetically extending the 3′ terminus of the DNA strand made in step j using the UP1 oligonucleotide as a template to make a functional double-stranded promoter using enzymatic in vitro nucleic acid synthesis, and
• m. transcribing multiple RNA transcripts from the functional promoter, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis.