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Nano-PCR: methods and devices for nucleic acid amplification and detection

An oligonucleotide and nucleotide sequence technology, applied in the field of nucleic acid amplification and detection, which can solve the problems of overall speed, efficiency, cost-effectiveness, and limitation of scope of use

Active Publication Date: 2008-03-26
NANOBIOTIX SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] Because of these and other factors, existing PCR-based techniques and detection systems are limited in overall speed, efficiency, cost-effectiveness, and scope of use

Method used

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  • Nano-PCR: methods and devices for nucleic acid amplification and detection
  • Nano-PCR: methods and devices for nucleic acid amplification and detection
  • Nano-PCR: methods and devices for nucleic acid amplification and detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1: Method and Apparatus Using Opposite Coated Surfaces

[0106] A pair of streptavidin-coated surfaces were fabricated according to standard methods (Sabanayagam, Smith, and Cantor. "Oligonucleotide immobilization on micropatterned streptavidin surfaces." Nucleic Acids Res. 2000, Vol.28, No.8 pp.i-iv ). Biotinylated dsDNA (biotinylated at both ends of one strand) was added to the above surfaces between which dsDNA was immobilized (Jeffrey M. Rothenberg and Meir Wilchek. p-Diazobenzoyl-biocytin: a new biotinylating reagent for DNA Nucleic Acids Research Volume 16 Number 14 1988). The surface density of DNA molecules can be controlled by adjusting the concentration of template applied to the surface. Tension greater than about 65 pN was exerted on the dsDNA by increasing the distance between the coated surfaces at room temperature. This denatures the dsDNA, leaving only the target ssDNA for amplification. The bound DNA is contacted with a primer containing a b...

Embodiment 2

[0108] Example 2: Methods and devices utilizing immobilized polymerases

[0109] Streptavidin-coated microchannel surfaces were fabricated according to standard methods (Sabanayagam, Smith, and Cantor. "Oligonucleotide immobilization on micropatterned streptavidin surfaces." Nucleic Acids Res. 2000, Vol.28, No.8 pp.i-iv ). Biotinylated DNA polymerase was flowed into the microchannel and incubated to saturate the surface. Commercial kits for enzyme biotinylation are available, eg, from Pierce Labs. Unbound enzyme flows out of the microchannel and target nucleotides (eg, ssDNA, RNA) and primers flow in at a chamber flow rate such that a force greater than 60 pN is applied to the nucleotides. Reduce the flow rate so that a force of 30pN to 60pN is applied to the nucleotides to form polymerase / nucleotide / primer complexes. The chamber flow rate was further reduced to less than 30 pN for primer extension. After completion of primer extension, the flow rate was increased to apply...

Embodiment 3

[0110] Embodiment 3: Utilize the method and device of DNA immobilization

[0111]Streptavidin-coated microchannel surfaces were fabricated according to standard methods. (Sabanayagam, Smith, and Cantor. "Oligonucleotide immobilization on micropatterned streptavidin surfaces." Nucleic Acids Res. 2000, Vol. 28, No. 8 pp. i-iv). Biotinylated dsDNA (biotinylated at one end of one strand) was flowed into a microchannel and incubated to allow surface binding (Jeffrey M. Rothenberg and Meir Wilchek. p-Diazobenzoyl-biocytin: a new biotinylating reagent for DNA Nucleic Acids Research Volume 16 Number 14 1988). Chamber fluid flow rates were established that exert a force greater than 65 pN on bound dsDNA. This denatures the dsDNA, leaving only the target ssDNA for amplification. Flow DNA polymerase, nucleotides, and biotinylated primers into the microchannel. The biotin reagent used can be purchased from commercial suppliers such as Molecular Probes or Pierce. For example, a biotin...

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PUM

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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 ore 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.

Description

technical field [0001] The invention relates to a nucleic acid amplification and detection method. In particular embodiments, the present invention discloses an improved method, device and material for polymerase chain reaction. Background technique [0002] Polymerase chain reaction (PCR) has become a common method for amplifying specific DNA sequences or RNA sequences. US Patent No. 4,683,202, issued to Mullis on July 28, 1987, and US Patent No. 4,683,195, issued to Mullis et al. on July 28, 1987, both disclose basic PCR methods. Since the advent of the PCR method, it has exerted a profound influence on the development of biotechnology and biomedicine. The disclosures of one or both of these patents have been cited in more than a thousand subsequent issued US patents. [0003] Generally, the DNA sequence amplification process needs to first select and obtain two oligonucleotide primers complementary to the ends of the target DNA sequence. Mix primers, polymerase, a mix...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34
CPCY02A50/30
Inventor 安妮塔·戈艾尔
Owner NANOBIOTIX SA
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