# US 2008/0305482 A1 · Methods and compositions for nucleic acid amplification

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- Number: US 2008/0305482 A1
- Document ID: US20080305482A1
- Status: Published application
- Kind: A1
- Application number: 11/962,072
- Filed: 2007-12-20
- Published: 2008-12-11
- Earliest claimed: 2006-12-21
- Assignees: Gen-Probe Incorporated
- Inventors: Steven T. BRENTANO; Dmitry LYAKHOV; James D. CARLSON; Norman C. NELSON
- Web page: https://patentgrep.com/patent/US20080305482A1

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6865: Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
- C12Q 2525/155: incorporating/generating a new priming site
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- C12Q 2525/197: incorporating a spacer/coupling moiety
- C12Q 2531/143: Promoter based amplification, e.g. NASBA, 3SR, TAS
- C12Q 2565/543: characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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.

## Claims

### Claim 1

A composition comprising:
a TSU promoter oligonucleotide that includes 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,
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, and
a means for directly or indirectly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide, thereby forming a target specific universal (TSU) primer complex.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the means for directly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide is a covalent linkage.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the covalent linkage is formed via a polynucleotide linker sequence.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the covalent linkage is formed via a non-nucleotide abasic linker compound.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the means for indirectly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide is a non-covalent linkage of members of a binding pair to join the TSU promoter oligonucleotide and the TSU non-promoter primer oligonucleotide to a support, wherein one member of the binding pair is present on the TSU promoter oligonucleotide or the TSU non-promoter primer oligonucleotide and the other member of the binding pair is attached to the support.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the means for directly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide is 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.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the means for indirectly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide is 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.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the S-oligonucleotide contains a first sequence complementary to the universal sequence in the TSU promoter oligonucleotide and the S-oligonucleotide contains a second sequence complementary to the universal sequence in the TSU non-promoter primer oligonucleotide.

### Claim 9 (depends on 1)

The composition of claim 1, further comprising a target specific capture oligonucleotide that contains a sequence that hybridizes specifically to a sequence in the target nucleic acid of the TSU promoter oligonucleotide and the TSU non-promoter primer at a sequence 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 support.

### Claim 10 (depends on 1)

The composition of claim 1, further comprising a universal promoter primer made up a 5′ promoter sequence and a 3′ universal sequence that is the same as the universal sequence of the TSU promoter oligonucleotide.

### Claim 11 (depends on 1)

The composition of claim 1, further comprising a universal primer made up a universal sequence that is the same as the universal sequence of the TSU non-promoter primer oligonucleotide.

### Claim 12 (depends on 1)

The composition 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.

### Claim 13 (depends on 7)

The composition of claim 7, wherein the S-oligonucleotide is made up of (1) a first terminal region sequence that is complementary to the U1 sequence of the TSU promoter primer and (2) a second terminal region sequence that is complementary to the U2 sequence of the TSU non-promoter primer, and (3) a linking moiety that links the first and second terminal region sequences.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the linking moiety is a non-nucleic acid chemical compound that covalently links the first and second terminal region sequences.

### Claim 15 (depends on 1)

The composition claim 1 further comprising at least one universal promoter primer made up of a 5′ promoter sequence and a 3′ U1 sequence and at least one target specific primer (TSP) made up of a sequence that is complementary to a sequence contained in an RNA transcript made from a double stranded DNA that contains a cDNA sequence made from synthetic extension of the 3′ end of the TSU promoter primer oligonucleotide.

### Claim 16

A method of amplifying a target nucleic acid comprising the steps of:
isolating a target nucleic acid from a mixture by binding to the target nucleic acid a target capture probe that binds specifically to the target nucleic acid and provides a means for attaching the bound target nucleic acid to a support that is separated from the mixture and further hybridizing to the target nucleic acid in the mixture a target specific universal (TSU) primer complex made up of
a TSU promoter primer oligonucleotide that includes 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,
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, and
a means for directly or indirectly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide,
hybridizing the TSU promoter primer oligonucleotide to a target sequence in the target nucleic acid via a TS sequence in the TSU promoter primer,
synthetically extending the 3′ terminus of the TSU promoter primer oligonucleotide 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,
hybridizing the TSU non-promoter primer oligonucleotide to the first cDNA strand by specific hybridization of the TS sequence in the TSU non-promoter primer oligonucleotide to a target sequence contained in the first cDNA strand,
synthetically extending the 3′ terminus of the TSU non-promoter primer oligonucleotide 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,
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 first target specific sequence (TS1), a second target specific sequence (TS2′), and a 3′ universal sequence (U2′) that is complementary to the U2 sequence,
hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcript at the U2′ sequence,
under isothermal conditions, synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand, and enzymatically removing the RNA transcript strand,
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,
under isothermal conditions, synthetically extending the 3′ terminus of the UP1 by enzymatic in vitro nucleic acid synthesis to made a dsDNA that contains a functional promoter, and
transcribing multiple RNA transcripts from the functional promoter of the dsDNA, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis under isothermal conditions by binding the UP2 primer and repeating the synthetic steps.

### Claim 17 (depends on 16)

The method of claim 16, further comprising the step of detecting the amplification products to indicate the presence of an analyte in the mixture from which the target nucleic acid was isolated.

### Claim 18

A method of amplifying a target nucleic acid comprising the steps of:
isolating a target nucleic acid from a mixture by binding to the target nucleic acid a target capture probe that binds specifically to the target nucleic acid and provides a means for attaching the bound target nucleic acid to a support that is separated from the mixture and further hybridizing to the target nucleic acid in the mixture a target specific universal (TSU) primer complex made up of
a TSU promoter oligonucleotide that includes 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 provider oligonucleotide that has a blocked 3′ terminus that is incapable of being extended by a polymerase,
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, and
a means for directly or indirectly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide,
hybridizing the TSU non-promoter primer oligonucleotide to a target sequence in the target nucleic acid via the TS sequence in the TSU non-promoter primer,
optionally hybridizing a blocker oligonucleotide with a 3′ blocked end that is incapable of being extended synthetically by a polymerase to a sequence on the target nucleic acid that is downstream from the position that the TSU non-promoter primer oligonucleotide hybridizes in the target nucleic acid,
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,
hybridizing the TSU promoter provider oligonucleotide to the first cDNA strand by specific hybridization of the TS sequence in the TSU promoter provider oligonucleotide to a target sequence contained in the first cDNA strand,
synthetically extending the 3′ terminus of the first cDNA by using sequence in the TSU promoter provider as a template to make a substantially double-stranded DNA that contains a functional promoter sequence and the U1 sequence,
enzymatically transcribing RNA transcripts from the functional promoter sequence to make RNA transcripts that contain a 5′ U1 region sequence, a first target specific sequence (TS1), a second target specific sequence (TS2′), and a 3′ universal sequence (U2′) that is complementary to the U2 sequence,
hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcript at the U2′ sequence,
under isothermal conditions, synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand, and enzymatically removing the RNA transcript strand,
hybridizing a universal promoter oligonucleotide (UP1) that contains a promoter sequence, a universal sequence U1, and a 3′ blocked end to the cDNA made in the previous step at the U1′ sequence,
under isothermal conditions, synthetically extending the 3′ terminus of the cDNA to make a functional double-stranded promoter by using the UP1 oligonucletide as a template and by enzymatic in vitro nucleic acid synthesis to made a dsDNA that contains a functional promoter, and
transcribing multiple RNA transcripts from the functional promoter of the dsDNA, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis under isothermal conditions by binding the UP2 primer and repeating the synthetic steps.

### Claim 19 (depends on 18)

The method of claim 18, further comprising the step of detecting the amplification products to indicate the presence of an analyte in the sample from which the target nucleic acid was isolated.

### Claim 20

A method of amplifying a target nucleic acid comprising the steps of:
isolating a target nucleic acid from a mixture by binding to the target nucleic acid a target capture probe that binds specifically to the target nucleic acid and provides a means for attaching the bound target nucleic acid to a support that is separated from the mixture and further hybridizing to the target nucleic acid in the mixture a target specific universal (TSU) promoter primer oligonucleotide that includes 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,
synthetically extending the 3′ terminus of the TSU promoter primer oligonucleotide 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,
adding to the amplification reaction mixture a target specific (TS) non-promoter primer that contains a second target specific sequence (TS2) which is different from the TS1,
hybridizing the TS non-promoter primer oligonucleotide to the first cDNA strand by specific hybridization of the TS2 sequence to a target sequence contained in the first cDNA strand,
synthetically extending the 3′ terminus of the TS non-promoter primer oligonucleotide 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,
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 first target specific sequence (TS1), a second target specific sequence (TS2′),
hybridizing a universal promoter primer oligonucleotide that contains a universal sequence U1′ to the RNA transcript at the U1 sequence,
under isothermal conditions, synthetically extending the 3′ terminus of the universal promoter primer by enzymatic in vitro nucleic acid synthesis to made a cDNA strand, and enzymatically removing the RNA transcript strand,
hybridizing a TS non-promoter primer oligonucleotide to a specific sequence in the cDNA made in the previous step,
under isothermal conditions, synthetically extending the 3′ terminus of the TS non-promoter primer by enzymatic in vitro nucleic acid synthesis to made a dsDNA that contains a functional promoter, and
transcribing multiple RNA transcripts from the functional promoter of the dsDNA, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis under isothermal conditions by repeating the synthetic steps.

### Claim 21 (depends on 20)

The method of claim 20, further comprising the step of detecting the amplification products to indicate the presence of an analyte in the mixture from which the target nucleic acid was isolated.

### Claim 22

A method of amplifying a target nucleic acid comprising the steps of:
isolating a target nucleic acid from a mixture by binding to the target nucleic acid a target capture probe that binds specifically to the target nucleic acid and provides a means for attaching the bound target nucleic acid to a support that is separated from the mixture and further hybridizing to the target nucleic acid in the mixture a TSU non-promoter primer oligonucleotide made up of a 5′ universal sequence (U2) and a 3′ target specific sequence (TS2),
hybridizing the TSU non-promoter primer oligonucleotide to a target sequence in the target nucleic acid via the TS2 sequence to a complementary sequence in the target nucleic acid,
hybridizing a blocker oligonucleotide with a 3′ blocked end that is incapable of being extended synthetically by a polymerase to a sequence on the target nucleic acid that is downstream from the position that the TSU non-promoter primer oligonucleotide hybridizes in the target nucleic acid,
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,
hybridizing to the first cDNA strand a target specific TS promoter provider oligonucleotide that includes a 5′ promoter sequence and a 3′ target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, and a blocked 3′ terminus that is incapable of being extended by a polymerase, by specific hybridization of the TS1 sequence to a complementary sequence in the first cDNA strand,
synthetically extending the 3′ terminus of the first cDNA by using sequence in the TS promoter provider as a template to make a substantially double-stranded DNA that contains a functional promoter sequence and a TS1 sequence,
enzymatically transcribing RNA transcripts from the functional promoter sequence to make RNA transcripts that contain a 5′ target specific sequence TS1, a target specific sequence TS2′ and a U2′ sequence,
hybridizing a universal primer oligonucleotide (UP2) that contains a universal sequence U2 to the RNA transcript at the U2′ sequence,
under isothermal conditions, synthetically extending the 3′ terminus of the UP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand, and enzymatically removing the RNA transcript strand,
hybridizing a TS promoter provider oligonucleotide that contains a promoter sequence and a 3′ blocked end to the cDNA made in the previous step,
under isothermal conditions, synthetically extending the 3′ terminus of the cDNA to make a functional double-stranded promoter by using the TS promoter provider oligonucletide as a template and by enzymatic in vitro nucleic acid synthesis to made a dsDNA that contains a functional promoter, and
transcribing multiple RNA transcripts from the functional promoter of the dsDNA, which transcripts are amplification products that may serve as templates for further enzymatic in vitro nucleic acid synthesis under isothermal conditions by repeating the synthetic steps.

### Claim 23 (depends on 22)

The method of claim 22, further comprising the step of detecting the amplification products to indicate the presence of an analyte in the sample from which the target nucleic acid was isolated.