Exponential base-greater-than-2 nucleic acid amplification
AssigneeCepheid
InventorRussell Higuchi
Life of this patent
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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.
Claims19
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 the double-stranded portion of the inner primer comprises combined sequence c-a annealed to a complementary combined sequence a′-c′, wherein combined sequence c-a, in double-stranded form, is more stable than combined sequence a-b, in double-stranded form, and wherein clamp sequence c is not capable of being copied during amplification.
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 the double-stranded portion of the inner primer comprises combined sequence c-a annealed to a complementary combined sequence a′-c′, wherein combined sequence c-a, in double-stranded form, is more stable than combined sequence a-b, in double-stranded form, and wherein clamp sequence c is not capable of being copied during amplification;
- 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.
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′, wherein the double-stranded portion of the first intermediate primer comprises combined sequence c1-d annealed to a complementary combined sequence d′-c1′, wherein combined sequence c1-d, in double-stranded form, is more stable than combined sequence d-a, in double-stranded form, and wherein clamp sequence c1 is not capable of being copied during amplification;
- 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 the double-stranded portion of the first inner primer comprises combined sequence c2-d-a annealed to a complementary combined sequence a′-d′-c2′, wherein combined sequence c2-d-a, in double-stranded form, is more stable than combined sequence d-a-b, in double-stranded form, and wherein clamp sequence c2 is not capable of being copied during amplification.
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′, wherein the double-stranded portion of the first intermediate primer comprises combined sequence c1-d annealed to a complementary combined sequence d′-c1′, wherein combined sequence c1-d, in double-stranded form, is more stable than combined sequence d-a, in double-stranded form, and wherein clamp sequence c1 is not capable of being copied during amplification;
- 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 the double-stranded portion of the first inner primer comprises combined sequence c2-d-a annealed to a complementary combined sequence a′-d′-c2′, wherein combined sequence c2-d-a, in double-stranded form, is more stable than combined sequence d-a-b, in double-stranded form, and wherein clamp sequence c2 is not capable of being copied during amplification;
- 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.
Dependencies are inferred from the claim text.