Method and apparatus for amplification of nucleic acid sequences by using thermal convection
First Claim
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1. A nucleic acid sequence amplification apparatus using PCR, which apparatus comprises:
- a plurality of heat sources which supply heat to, or remove heat from a plurality of specific regions in a sample,wherein the plurality of heat sources are arranged to maintain a spatial temperature distribution in the sample such that a first heat source providing heat to a lower portion of the sample is located lower in height than a second heat source removing heat from an upper portion of the sample and a relatively high temperature region is located lower in height than a relatively low temperature region in the sample,wherein the spatial temperature distribution comprises spatial regions fulfilling temperature conditions suitable for (i) a denaturation step in which double strand DNAs become separated to single strand DNAs, (ii) an annealing step in which the single strand DNAs formed in the denaturation step hybridize to the primers to form DNA-primer complexes, or (iii) a polymerization step in which the primers in the DNA-primer complexes are extended by the polymerization reaction,and wherein the apparatus further comprises an insulator positioned between the first and second heat sources.
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Abstract
The present invention provides a nucleic acid sequence amplification method and apparatuses thereof that are simple in the design and easy to miniaturize and integrate into complex apparatuses, with capability of using DNA polymerases that are not thermostable. In the present invention, a plurality of heat sources are combined to supply heat to, or remove heat from specific regions of the sample such that a specific spatial temperature distribution is maintained inside the sample by locating a relatively high temperature region lower in height than a relatively low temperature region.
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Citations
55 Claims
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1. A nucleic acid sequence amplification apparatus using PCR, which apparatus comprises:
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a plurality of heat sources which supply heat to, or remove heat from a plurality of specific regions in a sample, wherein the plurality of heat sources are arranged to maintain a spatial temperature distribution in the sample such that a first heat source providing heat to a lower portion of the sample is located lower in height than a second heat source removing heat from an upper portion of the sample and a relatively high temperature region is located lower in height than a relatively low temperature region in the sample, wherein the spatial temperature distribution comprises spatial regions fulfilling temperature conditions suitable for (i) a denaturation step in which double strand DNAs become separated to single strand DNAs, (ii) an annealing step in which the single strand DNAs formed in the denaturation step hybridize to the primers to form DNA-primer complexes, or (iii) a polymerization step in which the primers in the DNA-primer complexes are extended by the polymerization reaction, and wherein the apparatus further comprises an insulator positioned between the first and second heat sources. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A nucleic acid sequence amplification apparatus using PCR, which apparatus comprises:
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a plurality of heat sources which supply heat to, or remove heat from a plurality of specific regions within an opening configured to receive a reaction vessel and defined by the plurality of heat sources and an insulator, wherein the plurality of heat sources are arranged to maintain a spatial temperature distribution within the opening such that a first heat source providing heat to a lower portion of the opening is located lower in height than a second heat source removing heat from an upper portion of the opening and a relatively high temperature region is located lower in height than a relatively low temperature region in the opening, wherein the spatial temperature distribution comprises spatial regions fulfilling temperature conditions suitable for (i) a denaturation step in which double strand DNAs become separated to single strand DNAs, (ii) an annealing step in which the single strand DNAs formed in the denaturation step hybridize to the primers to form DNA-primer complexes, or (iii) a polymerization step in which the primers in the DNA-primer complexes are extended by the polymerization reaction, the insulator being positioned between the first and second heat sources and in contact with the opening, and further wherein at least one of the heat sources comprises a heating unit and a cooling unit. - View Dependent Claims (29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 53, 54, 55)
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30. A nucleic acid sequence amplification apparatus using PCR, which apparatus comprises:
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a plurality of heat sources which supply heat to, or remove heat from a plurality of specific regions in a sample, wherein the plurality of heat sources are arranged to maintain a spatial temperature distribution in the sample such that a first heat source providing heat to a lower portion of the sample is located lower in height than a second heat source removing heat from an upper portion of the sample and a relatively high temperature region is located lower in height than a relatively low temperature region in the sample, wherein the spatial temperature distribution comprises spatial regions fulfilling temperature conditions suitable for (i) a denaturation step in which double strand DNAs become separated to single strand DNAs, (ii) an annealing step in which the single strand DNAs formed in the denaturation step hybridize to the primers to form DNA-primer complexes, or (iii) a polymerization step in which the primers in the DNA-primer complexes are extended by the polymerization reaction, and further wherein at least one of the heat sources comprises a heating unit and a cooling unit. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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Specification