# US 11,028,434 B2 · Exponential base-greater-than-2 nucleic acid amplification

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- Number: US 11,028,434 B2
- Document ID: US11028434B2
- Status: Granted
- Kind: B2
- Application number: 16/359,947
- Filed: 2019-03-20
- Published: 2019-07-04
- Granted: 2021-06-08
- Earliest claimed: 2014-12-15
- Assignees: Cepheid
- Applicants: Cepheid
- Inventors: Russell Higuchi
- Web page: https://patentgrep.com/patent/US11028434B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2525/155: incorporating/generating a new priming site
- C12Q 2525/161: incorporating target specific and non-target specific sites
- C12Q 2525/191: incorporating an adaptor
- C12Q 2531/119: Strand displacement amplification [SDA]
- 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

Described herein are methods and compositions that provide highly efficient nucleic acid amplification. In some embodiments, this allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR.

## Claims

### Claim 1

A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprising oligonucleotides in the form of, or capable of forming, at least two first primers capable of hybridizing to the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer,
the first outer primer comprising a primer sequence a that specifically hybridizes to first template strand sequence a′; and
the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and
a clamp sequence c adjacent to, and 5′ of, primer sequence a, wherein clamp sequence c is not complementary to a first strand template sequence d′, which is adjacent to, and 3′ of, first strand template sequence a′, wherein combined sequence a-b contains more destabilizing bases than combined sequence c-a.

### Claim 2 (depends on 1)

The primer set of claim 1, wherein the primer set additionally comprises at least one second primer capable of specifically hybridizing to the second template strand.

### Claim 3

A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising:
(a) contacting the sample with:
(i) at least two first primers capable of hybridizing to the first template strand, wherein the at least two first primers comprise a first outer primer and a first inner primer,
the first outer primer comprising a primer sequence a that specifically hybridizes to first template strand sequence a′; and
the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b; and
a clamp sequence c adjacent to, and 5′ of, primer sequence a, wherein clamp sequence c is not complementary to a first strand template sequence d′, which is adjacent to, and 3′ of, first strand template sequence a′, wherein combined sequence a-b contains more destabilizing bases than combined sequence c-a; and
(ii) at least one second primer capable of specifically hybridizing to the second template strand,
wherein the contacting is carried out under conditions wherein the primers anneal to their template strands, if present; and
(b) amplifying the target nucleic acid, if present, using a DNA polymerase lacking 5′-3′ exonuclease activity, under conditions where strand displacement occurs, to produce amplicons that comprise sequence extending from template sequence a′ to the binding site for the second primer.

### Claim 4 (depends on 1)

The primer set of claim 1, wherein the DNA polymerase comprises strand displacement activity.

### Claim 5 (depends on 1)

The primer set of claim 1, wherein the Tm of combined sequence c-a, in double-stranded form, is greater than that of combined sequence a-b, in double stranded form.

### Claim 6 (depends on 1)

The primer set of claim 1, wherein combined sequence c-a is more GC-rich than combined sequence a-b, and/or contains more stabilizing bases.

### Claim 7 (depends on 1)

The primer set of claim 1, wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least two second primers capable of hybridizing to the second template strand, wherein the at least two second primers comprise a second outer primer and a second inner primer,
the second outer primer comprising a primer sequence e that specifically hybridizes to second template strand sequence e′; and
the second inner primer comprising a single-stranded primer sequence f that specifically hybridizes to second template strand sequence f′, wherein f′ is adjacent to, and 5′ of, e′, and wherein single-stranded primer sequence f is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence e adjacent to, and 5′ of, single-stranded primer sequence f; and
a clamp sequence g adjacent to, and 5- of, primer sequence e, wherein clamp sequence g is not complementary to second strand template sequence h′, which is adjacent to, and 3′, of second strand template sequence e′, wherein combined sequence e-f contains more destabilizing bases than combined sequence g-e.

### Claim 8 (depends on 7)

The primer set of claim 7, wherein the Tm of combined sequence g-e, in double-stranded form is greater than that of combined sequence e-f, in double-stranded form.

### Claim 9 (depends on 7)

The primer set of claim 7, wherein combined sequence g-e is more GC-rich than combined sequence e-f, and/or contains more stabilizing bases.

### Claim 10

A nucleic acid primer set for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the primer set comprising oligonucleotides in the form of, or capable of forming, at least three first primers capable of hybridizing to the first template strand, wherein the at least three first primers comprise a first outer primer, a first intermediate primer, and a first inner primer,
the first outer primer comprising a primer sequence d that specifically hybridizes to first template strand sequence d′;
the first intermediate primer comprising a primer sequence a that specifically hybridizes to first template strand sequence a′, wherein a′ is adjacent to, and 5′ of, d′, and wherein single-stranded primer sequence a is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence d adjacent to, and 5′ of, single-stranded primer sequence a; and
a clamp sequence c1 adjacent to, and 5- of, primer sequence d, wherein clamp sequence c1 is not complementary to a first strand template sequence i′, which is adjacent to, and 3′ of, first strand template sequence d′; and
the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b;
a primer sequence d adjacent to, and 5′ of, primer sequence a; and
a clamp sequence c2 adjacent to, and 5′ of, primer sequence d, wherein clamp sequence c2 is not complementary to first strand template sequence i′, wherein c1 has a different sequence than c2.

### Claim 11

A method for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the method comprising:
(a) contacting the sample with:
(i) at least three first primers capable of hybridizing to the first template strand, wherein the at least three first primers comprise a first outer primer, a first intermediate primer, and a first inner primer,
the first outer primer comprising a primer sequence d that specifically hybridizes to first template strand sequence d′;
the first intermediate primer comprising a primer sequence a that specifically hybridizes to first template strand sequence a′, wherein a′ is adjacent to, and 5′ of, d′, and wherein single-stranded primer sequence a is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence d adjacent to, and 5′ of, single-stranded primer sequence a; and
a clamp sequence c1 adjacent to, and 5- of, primer sequence d, wherein clamp sequence c1 is not complementary to a first strand template sequence i′, which is adjacent to, and 3′ of, first strand template sequence d′; and
the first inner primer comprising a single-stranded primer sequence b that specifically hybridizes to first template strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and wherein single-stranded primer sequence b is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence a adjacent to, and 5′ of, single-stranded primer sequence b;
a primer sequence d adjacent to, and 5′ of, primer sequence a; and
a clamp sequence c2 adjacent to, and 5′ of, primer sequence d, wherein clamp sequence c2 is not complementary to first strand template sequence i′, wherein c1 has a different sequence than c2; and
(ii) at least one second primer capable of specifically hybridizing to the second template strand,
wherein the contacting is carried out under conditions wherein the primers anneal to their template strands, if present; and
(b) amplifying the target nucleic acid, if present, using a DNA polymerase lacking 5′-3′ exonuclease activity, under conditions where strand displacement occurs, to produce amplicons that comprise sequence extending from template sequence a′ to the binding site for the second primer.

### Claim 12 (depends on 11)

The method of claim 11, wherein the DNA polymerase comprises strand displacement activity.

### Claim 13 (depends on 10)

The primer set of claim 10, wherein the Tm of combined sequence c1-d, in double-stranded form, is greater than that of combined sequence d-a, in double-stranded form, and the Tm of combined sequence c2-d-a, in double-stranded form, is greater than that of combined sequence d-a-b, in double-stranded form.

### Claim 14 (depends on 10)

The primer set of claim 10, wherein combined sequence c1-d is more GC-rich than combined sequence d-a, and/or contains more stabilizing bases, and combined sequence c2-d-a is more GC-rich than combined sequence d-a-b, and/or contains more stabilizing bases than combined sequence d-a-b.

### Claim 15 (depends on 11)

The method of claim 11, wherein said amplifying amplifies the target nucleic acid at the rate of up to 4number of cycles during the exponential phase of PCR.

### Claim 16 (depends on 11)

The method of claim 11, wherein said amplifying permits detection of a single copy nucleic acid in a biological sample within about 25%-55% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.

### Claim 17 (depends on 10)

The primer set of claim 10, wherein the second primer comprises oligonucleotides in the form of, or capable of forming, at least three second primers capable of hybridizing to the second template strand, wherein the at least three second primers comprise a second outer primer, a second intermediate primer, and a second inner primer,
the second outer primer comprising a primer sequence h that specifically hybridizes to second template strand sequence h′;
the second intermediate primer comprising a single-stranded primer sequence e that specifically hybridizes to second template strand sequence e′, wherein e′ is adjacent to, and 5′ of, h′, and wherein single-stranded primer sequence e is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence h adjacent to, and 5′ of, single-stranded primer sequence e; and
a clamp sequence g1 adjacent to, and 5′ of, primer sequence h, wherein clamp sequence g1 is not complementary to a second strand template sequence j′, which is adjacent to, and 3′, of second strand template sequence h′; and
the second inner primer comprising a single-stranded primer sequence f that specifically hybridizes to first template strand sequence f′, wherein f′ is adjacent to, and 5′ of, e′, and wherein single-stranded primer sequence f is linked at its 5′ end to a first strand of a double-stranded primer sequence comprising:
a primer sequence e adjacent to, and 5′ of, single-stranded primer sequence f;
a primer sequence h adjacent to, and 5′ of, primer sequence e; and
a clamp sequence g2 adjacent to, and 5′ of, primer sequence h, wherein clamp sequence c2 is not complementary to first strand template sequence j′.

### Claim 18 (depends on 17)

The primer set of claim 17, wherein the Tm of combined sequence g1-h, in double-stranded form, is greater than that of combined sequence h-e, in double-stranded form, and the Tm of combined sequence g2-h-e, in double-stranded form, is greater than that of combined sequence h-e-f, in double-stranded form.

### Claim 19 (depends on 17)

The primer set of claim 17, wherein combined sequence g1-h is more GC-rich than combined sequence h-e, and/or contains more stabilizing bases, and combined sequence g2-h-e is more GC-rich than combined sequence h-e-f, and/or contains more stabilizing bases than combined sequence h-e-f.

### Claim 20 (depends on 12)

The method of claim 12, wherein said amplifying amplifies the target nucleic acid at the rate of up to 8number of cycles during the exponential phase of PCR.

### Claim 21 (depends on 12)

The method of claim 12, wherein said amplifying permits detection of a single copy nucleic acid in a biological sample within about 42%-72% fewer amplification cycles than would be required for said detection using only a single forward and a single reverse primer.

### Claim 22 (depends on 17)

The primer set of claim 17, wherein clamp sequences c1, c2, g1, and g2 are not capable of being copied during amplification.

### Claim 23 (depends on 22)

The primer set of claim 22, wherein clamp sequences c1, c2, g1, and g2 each comprise 2′-O-methyl RNA.

### Claim 24 (depends on 17)

The primer set of claim 17, wherein:
the double-stranded primer sequence of the first inner primer and the first intermediate primer; and/or
the second inner primer and the second intermediate primer, if present,
does/do not comprise a hairpin sequence.

### Claim 25 (depends on 17)

The primer set of claim 17, wherein:
the double-stranded primer sequence of the first inner primer comprises a hairpin sequence in which clamp sequence c2 is linked to complementary sequence c2′; and/or
the double-stranded primer sequence of the first intermediate primer comprises a hairpin sequence in which clamp sequence c1 is linked to complementary sequence c1′; and/or
the double-stranded primer sequence of the second inner primer, if present, comprises a hairpin sequence in which clamp sequence g2 is linked to complementary sequence g2′; and/or
the double-stranded primer sequence of the second intermediate primer, if present, comprises a hairpin sequence in which clamp sequence g1 is linked to complementary sequence g1′.

### Claim 26 (depends on 10)

The primer set of claim 10, wherein combined sequence d-a contains more destabilizing bases than combined sequence c1-d, and/or combined sequence d-a-b contains more destabilizing bases than combined sequence c2-d-a.

### Claim 27 (depends on 17)

The primer set of claim 17, wherein combined sequence h-e contains more destabilizing bases than combined sequence g1-h, and/or combined sequence h-e-f contains more destabilizing bases than combined sequence g2-h-e.