Nano-PCR: methods and devices for nucleic acid amplification and detection
First Claim
1. A method for amplifying a nucleotide sequence wherein the method comprises:
- (a) contacting one or more template strands of single-stranded DNA with one or more oligonucleotide primers complementary to a portion of each template strand and;
(b) permitting hybridization of at least one primer to the complimentary portion of the template strands;
(c) contacting the template strands with a DNA polymerase and at least four different nucleoside triphosphates,(d) permitting extension of the primers by the DNA polymerase thereby forming extension products bound to the template strands;
(e) separating the extension products from the template strands; and
thereafter repeating steps (a), (b), (c), and (d) one or more times with step (e) performed before each repetition;
wherein permitting hybridization of at least one primer to the complimentary portion of the template strands comprises, controlling the hybridization by controlling an application of stress to the template strands, orwherein permitting extension of the primers by the DNA polymerase comprises controlling the extension of the primers by the DNA polymerase to form extension products complementary to each template strands by controlling an application of stress to the template strands, orwherein separating the extension products from the template strands comprises separating the extension products from the template strands by applying stress to the template strands,or a combination of these wherein stress is applied to the template strands during more than one of steps (b), (d), and (e).
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Accused Products
Abstract
Methods, devices, and compositions are described that provide for amplification of nucleic acid sequences without reliance upon temperature cycling, thus freeing the methods from conventional benchtop thermal cycling devices. Denaturation of double stranded nucleic acids, primer annealing, and precision control over primer extension by polymerase can be accomplished by applying stress to a nucleic acid. These methods can provide one or more benefits over conventional PCR methods including: precision control over the PCR process; generally improved fidelity; improved accuracy over problematic sequences such as GC-rich or tandem repeat regions; greater sequence length; increased reaction yield; reduced experimental time; greater efficiency; lower cost; greater portability; and, robustness to various environmental parameters, such as temperature, pH, and ionic strengths.
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Citations
1 Claim
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1. A method for amplifying a nucleotide sequence wherein the method comprises:
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(a) contacting one or more template strands of single-stranded DNA with one or more oligonucleotide primers complementary to a portion of each template strand and; (b) permitting hybridization of at least one primer to the complimentary portion of the template strands; (c) contacting the template strands with a DNA polymerase and at least four different nucleoside triphosphates, (d) permitting extension of the primers by the DNA polymerase thereby forming extension products bound to the template strands; (e) separating the extension products from the template strands; and
thereafter repeating steps (a), (b), (c), and (d) one or more times with step (e) performed before each repetition;wherein permitting hybridization of at least one primer to the complimentary portion of the template strands comprises, controlling the hybridization by controlling an application of stress to the template strands, or wherein permitting extension of the primers by the DNA polymerase comprises controlling the extension of the primers by the DNA polymerase to form extension products complementary to each template strands by controlling an application of stress to the template strands, or wherein separating the extension products from the template strands comprises separating the extension products from the template strands by applying stress to the template strands, or a combination of these wherein stress is applied to the template strands during more than one of steps (b), (d), and (e).
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Specification