Patent ID: 11952621
Assignee: CEPHEID
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 18:
19. 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.