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Assignees
Member
CepheidCepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Cepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Publication Number
US-11952621-B2
Publication Date
2024-04-09
Expiration Date
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.
Core Innovation
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.
PCR in general has several limitations. PCR amplification can only achieve less than two fold increase of the amount of target sequence at each cycle, it is still relatively slow, and the sensitivity of this method is typically limited, making it difficult to detect target that may be present at only a few molecules in a single reaction.
Described herein are methods and compositions based on the use of novel primers (e.g., novel inner primers) designed to so that the outer primer binding site is maintained in the amplicons produced upon amplification. A solution to this problem is to use an additional 5′ add-on to the inner primer (i.e., a sequence in addition to the outer primer sequence) together with an oligonucleotide complementary to both sequences, which is referred to herein as a "clamp."
Claims Coverage
Four independent claims were identified and five main inventive features were extracted from those claims.
Primer set comprising first outer and inner primers
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′.
Double-stranded portion c-a more stable than a-b and clamp not copyable
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.
Method using primers and strand displacement polymerase lacking 5′-3′ exonuclease activity
contacting the sample with the primers as recited and 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.
Three-primer set comprising outer, intermediate, and inner primers with clamps
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′; 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.
Method using three-primer set and strand displacement polymerase lacking 5′-3′ exonuclease activity
contacting the sample with the three-primer set as recited and 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.
The independent claims disclose primer set architectures in which inner/intermediate primers include a double-stranded clamp-anchored region (c, c1, c2) that is more stable than the adjacent primer region (a-b, d-a, d-a-b) and clamps that are not capable of being copied, together with methods that employ a DNA polymerase lacking 5′-3′ exonuclease activity under strand displacement conditions to produce amplicons extending from template sequence a′ to the second primer binding site.
Stated Advantages
Highly efficient nucleic acid amplification allowing a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR.
Sustained higher per-cycle amplification rates that reduce the number of amplification cycles required to detect a single-copy nucleic acid, with stated reductions of about 12%-42% (hemi-nested two-primer), about 36%-66% (fully-nested two-primer), about 25%-55% (hemi-nested three-primer), and about 42%-72% (fully-nested three-primer).
Documented Applications
Sensitive diagnostic assays based on nucleic acid detection.
Detection of nucleic acids associated with pathogens such as viruses, bacteria, protozoa, or fungi; detection of RNAs indicative of disease; analysis of genomic DNA for polymorphisms, alleles, or haplotypes; genotyping; and applications including forensic or paternity determinations.
Detecting and optionally quantifying target nucleic acids, including detection of a single copy nucleic acid in a biological sample and analysis of nucleic acids from a single cell.
Use with automated sample handling and analysis platforms, exemplified by the GeneXpert® system.
Kits comprising one or more reagents and instructions for carrying out the described amplification methods.
Analysis of nucleic acids from a variety of biological sample types including cells, bodily fluids (e.g., blood, a blood fraction, urine), tissue samples, plasma, serum, spinal fluid, lymph fluid, peritoneal fluid, pleural fluid, oral fluid, respiratory, intestinal, genital, or urinary tract samples, tears, saliva, blood cells, stem cells, tumors, paraffin-embedded tissue samples, and needle biopsies.
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