Exponential base-3 and greater nucleic acid amplification with reduced amplification time
Inventors
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Assignees
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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-12098368-B2
Publication Date
2024-09-24
Expiration Date
Abstract
Described herein are methods and compositions that provide highly efficient nucleic acid amplification. In some embodiments, this allows a 3-fold or greater increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Modified bases can be employed in primers to provide this base-3 or greater amplification with satisfactory PCR cycle times, which are improved, as compared to those observed in the absence of modified bases.
Core Innovation
Described herein are methods and compositions that provide highly efficient nucleic acid amplification, including primer sets and associated methods that allow a 3-fold or greater increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. The invention employs novel primers (e.g., novel inner primers) designed so that the outer primer binding site is maintained in the amplicons produced upon amplification, and modified bases can be employed in primers to provide this base-3 or greater amplification with satisfactory PCR cycle times.
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 relatively slow, and the sensitivity is typically limited, making it difficult to detect target that may be present at only a few molecules in a single reaction. The present methods and compositions address these limitations by increasing amplification efficiency and reducing amplification time.
The approaches include primer architectures in which inner and intermediate primers comprise single-stranded primer portions linked to first strands of double-stranded primer sequences that include primer sequence(s) and a clamp sequence not complementary to adjacent template sequence, wherein combined double-stranded primer sequences (e.g., c-a, c1-d, c2-d-a, g-e, g1-h, g2-h-e) are designed to be more stable than adjacent combined sequences (e.g., a-b, d-a, d-a-b, e-f, h-e, h-e-f), and wherein modified bases are incorporated so that modified bases preferentially pair with their unmodified forms rather than with each other. Amplification is carried out 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, enabling sustained greater-than-two-fold amplification per cycle and reduced cycle number and cycle time.
Claims Coverage
Two independent claims identified. Main inventive features extracted from each independent claim are listed below.
Three-primer set architecture with double-stranded primer portions and clamps
A primer set comprising at least three first primers that hybridize to a first template strand (first outer, first intermediate, first inner), wherein inner and intermediate primers include single-stranded primer sequences linked at their 5′ ends to first strands of double-stranded primer sequences that include primer sequence d and clamp sequences c1 or c2, and wherein clamp sequence c1 or c2 is not complementary to adjacent first template strand sequence i′.
Matched three-primer set on complementary strand
The primer set additionally comprises at least one second primer capable of hybridizing to the second template strand, the second primer set comprising at least three second primers (second outer, second intermediate, second inner) with analogous double-stranded primer sequences and clamp sequences g1 and g2 that are not complementary to adjacent second template strand sequence j′.
Double-stranded combined sequences complementary to template-derived complementary strands
Combined double-stranded primer sequences (e.g., c1-d and c2-d-a; g1-h and g2-h-e) have corresponding second strands (e.g., c1′-d′ and c2′-d′-a′; g1′-h′ and g2′-h′-e′) that are complementary to the combined primer sequences, such that combined sequence c1′-d′ is complementary to c1-d and combined sequence c2′-d′-a′ is complementary to c2-d-a, etc.
Use of modified bases with preferential pairing to unmodified forms
One or more primer sequences (outer, intermediate, inner and corresponding complementary strands) comprise modified bases wherein the unmodified forms of paired modified bases are complementary, and the modified bases preferentially pair with the unmodified forms, as compared to pairing between the modified bases themselves (e.g., first/third and second/fourth, fifth/seventh and sixth/eighth relationships).
The independent composition claim covers primer sets with three-primer nested architectures on one or both template strands featuring double-stranded primer portions with non-template-complementary clamp sequences and incorporation of modified bases that preferentially pair with unmodified complements; these features together define the claimed primer set.
Stated Advantages
Achieves a 3-fold or greater increase of amplification product for each amplification cycle, providing increased sensitivity and speed over conventional PCR.
Sustains greater-than-two-fold amplification per cycle to reduce the number of amplification cycles required to detect a single-copy nucleic acid (examples include reductions of about 12%-42% for hemi-nested two-primer sets, about 36%-66% for fully-nested two-primer sets, about 25%-55% for hemi-nested three-primer sets, and about 42%-72% for fully-nested three-primer sets).
Use of modified bases in primers reduces the time required to complete each amplification cycle, with claimed reductions ranging from 10%-95% in various embodiments and a specific claimed range of 50%-85% in some embodiments.
Documented Applications
Sensitive diagnostic assays based on nucleic acid detection, including detection and optional quantification of target nucleic acids.
Detection of nucleic acids associated with pathogens (viruses, bacteria, protozoa, fungi), RNAs indicative of disease or tissue-specific expression, genomic DNA for polymorphisms (e.g., SNPs), alleles, haplotypes, and sequences altered in genetic diseases or other pathologies.
Detection of single-copy nucleic acid in biological samples and amplification from nucleic acids obtained from a single cell.
Use in automated sample handling and analysis platforms exemplified by the GeneXpert® system, including cartridge-based sample extraction, amplification, and detection.
Provision in kits comprising one or more reagents and instructions for carrying out the disclosed amplification methods.
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