Isothermal amplification components and processes
Inventors
Miller, Andrew P. • Zhang, Honghua
Assignees
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Publication Number
US-9617587-B1
Publication Date
2017-04-11
Expiration Date
Abstract
The technology relates in part to methods and compositions for isothermal amplification of nucleic acids.
Core Innovation
The invention relates to isothermal nucleic-acid amplification and detection for determining the presence, absence or amount of a target sequence in sample nucleic acid. Amplification is performed by contacting non-denatured sample nucleic acid under cleavage agent-free, helicase-free and recombinase-free isothermal amplification conditions, and the target sequence comprises a first strand and a second strand that are complementary to each other.
The method uses a first oligonucleotide and a second oligonucleotide that are continuously complementary to sequences in the first and second strands, respectively. A hyperthermophile polymerase activity component is contacted with the oligonucleotides and the sample nucleic acid to generate a nucleic acid amplification product consisting of first and second nucleotide sequences continuously complementary to, or substantially identical to, the first and second polynucleotide sequences, with a spacer sequence comprising 1 to 10 bases flanked by the first and second nucleotide sequences.
Detection is performed using a real-time detection method, and the detecting step is performed in 10 minutes or less from the time the sample nucleic acid is contacted. The approach includes real-time detection approaches such as fluorescent signal and molecular beacon.
Claims Coverage
The document includes one independent claim, directed to a complete workflow for determining presence, absence or amount of a target sequence using cleavage agent-free, helicase-free and recombinase-free isothermal amplification with a hyperthermophile polymerase and a characteristic amplification product containing a 1 to 10 base spacer, followed by real-time detection in 10 minutes or less. Dependent claims further constrain reaction conditions, component properties, spacer length, spacer identity relationships, and detection modalities.
Cleavage agent-free, helicase-free, recombinase-free isothermal amplification of complementary strands
amplifying a target sequence in the sample nucleic acid by contacting non-denatured sample nucleic acid under cleavage agent-free, helicase-free and recombinase-free isothermal amplification conditions, wherein the target sequence comprises a first strand and a second strand that are complementary to each other
Continuously complementary first and second oligonucleotides generating a spacer-flanked amplification product
contacting a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide consists of a first polynucleotide continuously complementary to a sequence in the first strand and the second oligonucleotide consists of a second polynucleotide continuously complementary to a sequence in the second strand, and generating a nucleic acid amplification product consisting of first and second nucleotide sequences continuously complementary to, or substantially identical to, the first and second polynucleotides, with a spacer sequence comprising 1 to 10 bases flanked by the first and second nucleotide sequences
Hyperthermophile polymerase activity for isothermal amplification without cleavage agents
providing at least one component providing a hyperthermophile polymerase activity, thereby generating the nucleic acid amplification product having the spacer sequence
Real-time detection within 10 minutes for determining presence, absence or amount
detecting the nucleic acid amplification product using a real-time detection method performed in 10 minutes or less from the time the sample nucleic acid is contacted with the first and second oligonucleotides and the hyperthermophile polymerase activity, whereby the presence, absence or amount of a target sequence is determined
Overall, the claim coverage centers on a helicase-free, recombinase-free, cleavage agent-free isothermal amplification scheme for complementary first and second strands using continuously complementary oligonucleotides, producing an amplification product with a 1 to 10 base spacer flanked by two nucleotide sequences corresponding to the primer polynucleotides, and determining presence, absence or amount via real-time detection completed in 10 minutes or less. Dependent claims further narrow example constraints such as constant temperature ranges, oligonucleotide length ranges, spacer length ranges, spacer identity relationships, and fluorescent signal-based detection.
Stated Advantages
Real-time detection performed in 10 minutes or less from the time the sample nucleic acid is contacted.
Avoids thermocycling and denaturation processes by using isothermal amplification conditions.
Operates under cleavage agent-free, helicase-free and recombinase-free conditions.
Documented Applications
Determining the presence, absence or amount of a target sequence in sample nucleic acid.
Real-time nucleic acid amplification with real-time fluorescence detection (including molecular beacon approaches) and confirmation using ESI-MS [procedural detail omitted for safety].
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