WO2014041093A1 - Matrice solide pour l'amplification d'acide nucléique en une étape - Google Patents

Matrice solide pour l'amplification d'acide nucléique en une étape Download PDF

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Publication number
WO2014041093A1
WO2014041093A1 PCT/EP2013/068947 EP2013068947W WO2014041093A1 WO 2014041093 A1 WO2014041093 A1 WO 2014041093A1 EP 2013068947 W EP2013068947 W EP 2013068947W WO 2014041093 A1 WO2014041093 A1 WO 2014041093A1
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Prior art keywords
nucleic acid
solid matrix
cyclodextrin
amplification
reagent
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PCT/EP2013/068947
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English (en)
Inventor
Michael Kenneth Kenrick
Simon Laurence John Stubbs
Cheryl Louise POTTS
Kathryn Louise LAMERTON
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Ge Healthcare Uk Limited
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Priority claimed from GBGB1216387.9A external-priority patent/GB201216387D0/en
Application filed by Ge Healthcare Uk Limited filed Critical Ge Healthcare Uk Limited
Priority to GB1502256.9A priority Critical patent/GB2519465B/en
Publication of WO2014041093A1 publication Critical patent/WO2014041093A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present invention relates to the field of nucleic acid amplification, particularly to the use of a polymerase chain reaction to amplify nucleic acid.
  • the invention provides methods and kits which can be used to amplify nucleic acid by embedding PCR reagents onto a solid matrix for easy amplification of DNA samples.
  • the invention has applications in the long term storage and easy processing of nucleic acid and is particularly useful in genotyping, diagnostics and forensics.
  • PCR polymerase chain reaction
  • EP 1563091 (Smith et al, Whatman) relates to methods for storing nucleic acids from samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media. Moreover, the document describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices in tubes, columns, or multiwell plates.
  • WO 90/003959 describes a cellulose-based solid support for the storage of DNA, including blood DNA, comprising a solid matrix having a compound or composition which protects against degradation of DNA incorporated into or absorbed on the matrix. This document also discloses methods for storage of DNA using the solid medium, and for recovery of or in situ use of DNA.
  • EP 2290099 B 1 (Qiagen) describes again a method for processing and amplifying DNA.
  • the method includes the steps of contacting the sample containing DNA to a solid support wherein a lysis reagent is bound to the solid support.
  • the DNA is subsequently treated with a DNA purifying reagent and is purified.
  • the application does not include a sequestrant on the solid support and requires a separate step for the removal of the lysis reagent and purification of the DNA before amplification.
  • WO 96/39813 discloses a solid medium for storing a sample of genetic material and subsequent analysis; the solid medium comprising a protein denaturing agent and a chelating agent.
  • the method described is for chelating agents which are any compound capable of complexing multivalent ions including Group II and Group III multivalent metal ions and transition metal ions.
  • the invention does not specifically mention cyclodextrin as a chelating agent, nor does it suggest the PCR analysis could be performed in a single step.
  • US 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant.
  • the advantage of this system is that conventional detergent removal requires a separation step however with the addition of cyclodextrin to neutralize the detergent it would remove the separation step needed and reduce chance of contamination.
  • GB 2346370 (Cambridge Molecular Technologies Ltd) discloses applying a sample comprising cells containing nucleic acid to a filter, the cells are retained by the filter and contaminants are not. The cells are lysed on the filter and retained alongside the nucleic acid. Subsequent steps filter out the cell lysate while retaining the nucleic acid.
  • WO 96/18731 (Deggerdal) describes a method of isolating nucleic acid whereby the sample is bound to a solid support and sample is contacted with a detergent and subsequent steps performed to isolate the nucleic acid. The method does not include a sequestrant being bound to the solid support.
  • WO 00/53807 discloses a medium for the storage and lysis of samples containing genetic material which can be eluted and analysed.
  • the medium is coated with a lysis reagent.
  • the medium could be coated with a weak base, a chelating agent, a surfactant and optionally uric acid.
  • WO 99/38962 (Health, Gentra Systems Inc.) describes a solid support with a bound lysis reagent.
  • the lysis reagent can comprise of a detergent, a chelating agent, water and optionally an R A digesting enzyme.
  • the solid support does not contain cyclodextrin and requires further steps for purification of the nucleic acid for
  • WO 02/40699 discloses a method for the analysis of nucleic acids by applying a sample on a matrix where the matrix comprises a weak base, a chelating agent and an anionic surfactant or detergent.
  • the suggested chelating agent was EDTA but was not limited to this embodiment.
  • the application does not suggest that cyclodextrin as the chelating agent.
  • WO 91/02040 describes an invention using cyclodextrin-labelled primers in an amplification reaction mixture for qualitative and quantitative nucleic acid sequence analysis.
  • the benefits were a higher signal efficiency and versatility in label colors.
  • WO 95/32739 discloses an oligonucleotide noncovalently complexed with a cyclodextrin.
  • cyclodextrin discloses an oligonucleotide noncovalently complexed with a cyclodextrin.
  • cyclodextrin discloses an oligonucleotide noncovalently complexed with a cyclodextrin.
  • cyclodextrin with oligonucleotides was for the cellular uptake of oligonucleotides and not for the amplification of nucleotides in a PCR reaction.
  • WO 2010/066908 (Beckers et al.,) describes the use of cyclodextrins to improve the specificity, sensitivity and/or yield of PCR.
  • the method claimed is an amplification reaction which is performed in a reaction mixture comprising at least one cyclodextrin and performing the amplification reaction on said reaction.
  • a solid matrix embedded with cyclodextrin for use in PCR amplification or a suggestion towards the combining of the solid matrix with cyclodextrin.
  • cyclodextrins can act as molecular chelating agents and consist of six a-cyclodextrin, seven ⁇ -cyclodextrin, eight ⁇ -cyclodextrin or more glucopyranose units linked by a-(l,4) bonds. Cyclodextrins form a hydrophilic outside which allows the molecule to dissolve in water and an apolar cavity that is hydrophobic. This cavity allows cyclodextrins to form an inclusion complex with appropriately sized non-polar moieties.
  • the height of the cavity is the same for all types of cyclodextrin types but internal diameter and volume is determined by the number of glucose units.
  • cyclodextrin would be considered a chelating agent.
  • Current methods for DNA amplification involve a DNA purification procedure which often involves several steps which increases the chance of contamination. This is a tedious process and prior art methods have a number of clear disadvantages in terms of cost, complexity and in particular, user time.
  • column-based nucleic acid purification is a typical solid phase extraction method to purify nucleic acids. This method relies on the nucleic acid binding through adsorption to silica or other support depending on the pH and the salt content of the buffer.
  • suitable buffers include Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray
  • nucleic acid purification on spin columns typically involves three time-consuming and complex steps/stages:
  • the sample containing nucleic acid is added to the column and the nucleic acid binds due to the lower pH (relative to the silanol groups on the column) and salt concentration of the binding solution, which may contain buffer, a denaturing agent (such as guanidine hydrochloride), Triton X-100, isopropanol and a pH indicator; the column is washed with 5 mM KP04 pH 8.0 or similar, 80% EtOH); and the column is eluted with buffer or water.
  • a denaturing agent such as guanidine hydrochloride
  • Triton X-100 Triton X-100
  • isopropanol isopropanol
  • a pH indicator a pH indicator
  • chaotropic agents such that DNA binds to silica or glass particles or glass beads. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions.
  • Typical chaotropic agents include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns.
  • the best defence against PCR amplification failure in forensics applications is to combine sound sample handling and processing techniques with extraction systems proven to efficiently purify DNA.
  • the present invention addresses this problem and provides methods and kits which can be used for single step amplification of nucleic acid from solid supports, particularly cellulose-derived supports.
  • the present invention provides methods and kits which can be used to store and amplify nucleic acid by embedding PCR reagents onto a solid matrix for easy
  • a solid matrix for storing and/or amplification of nucleic acid comprising a lysis reagent and a sequestering reagent.
  • the advantage of incorporating the lysis reagent and sequestrant on to the solid matrix is to reduce the number of steps required for nucleic acid
  • nucleic acid is selected from the group consisting of DNA, RNA and oligonucleotide.
  • nucleic acid is used herein synonymously with the term “nucleotides” and includes DNA, such as plasmid DNA and genomic DNA; RNA, such as mRNA, tRNA, sRNA and RNAi; and protein nucleic acid, PNA.
  • the lysis reagent comprises an anionic surfactant or detergent.
  • Sodium dodecyl sulphate (SDS) is an example of an anionic surfactant frequently used to lyse biological cells.
  • the sequestering agent is a cyclodextrin.
  • the cyclodextrin may be selected from a group consisting of a-cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin and derivatives thereof.
  • the sequestrant is a-cyclodextrin.
  • the cyclodextrin may be selected from the group consisting of 6-0-a-D-Maltosyl-P cyclodextrin, hydroxyethyl- ⁇ - cyclodextrin, hydroxypropyl-P-cyclodextrin and 2-hydroxypropyl-P-cyclodextrin.
  • the sequestering reagent is not a chelating agent.
  • a chelating agent is a chemical compound that combines with a metal to form a chelate, often used to trap heavy metal ions (Colins English Dictionary, ⁇ HarperCollins Publishers 2003).
  • One example of a lysis reagent is sodium dodecyl suphate; sodium is a metal ion however according to Ramamurthy Palepu and Vincent C. Reinsborough (Can J. Chem Vol 66, 325-328, 1988) it is the hydrophobic tail of the molecule that interacts with the cyclodextrin not the hydrophilic head.
  • the solid matrix is selected from one of the following: glass, glass fiber, glass micro fiber, silica, silica gel, silica oxide, cellulose, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool or porous ceramics.
  • the solid matrix may comprise a glass or silica-based solid phase medium, a plastics-based solid phase medium or a cellulose-based solid phase medium.
  • the solid support is preferably a cellulose-based matrix. Examples of cellulose-based matrices include FTATM, 903 neonatal cards and 31-ETF cards available from GE Healthcare.
  • the cellulose based matrix is in the form of a pre punched disc.
  • the cellulose based matrix can also be in the form of an FTA pre punched disc.
  • a solid matrix for storing and/or amplification of nucleic acid comprising a lysis reagent and a sequestering reagent whereby the sequestering reagent and the lysis reagent are separated physically in the matrix.
  • the lysis reagent could also be adjacent to the sequestering reagent on said solid matrix.
  • a spacer can be used to separate the sequestering and lysis reagent.
  • the solid matrix further comprises a lyophilised or impregnated or printed Ready to Go PCR reagent for one step nucleic acid amplification wherein the Ready to Go PCR beads comprise of a DNA polymerase, deoxyribonucleotide triphosphate (dNTP), a reaction buffer and at least one primer.
  • dNTP deoxyribonucleotide triphosphate
  • the advantage of dried or lyophilised formulations impregnated or printed onto to a solid matrix is that they can be easily solublised by the addition of water, thus saving operator time and facilitating operator usage.
  • the lysis reagent (20), sequestrant (40) and PCR reagents (50) are physically separated by a spacer (30) as demonstrated in Figure 1 A.
  • the lysis reagent (70), sequestrant (90) and PCR reagents (100) are physically separated by a spacer (80) as demonstrated in Figure IB.
  • a method for amplification of nucleic acid using the polymerase chain reaction comprising the steps:
  • the method of the invention can be used either in single tube or a high-throughput 96-well format in combination with automated sample processing as described by Baron et al, (2011, Forensics Science International: Genetics Supplement Series, 93, e560- e561). This approach would involve a minimal number of steps and increase sample throughput. The risk of operator-induced error, such as cross-contamination is also reduced since this procedure requires fewer manipulations compared to protocols associated with currently used, more labour intensive kits (e.g. QIAmp DNA blood mini kit, Qiagen). The risk of sample mix-up is also reduced since the procedure requires few manipulations. Importantly, the method is readily transferable to a multi-well format for high-throughput screening. The present invention can thus improve sample storage and processing for carrying out PCR reactions to aid genetic interrogations.
  • the invention can be conducted in a 96 well/high throughput format to facilitate sample handling and thus eliminate batch processing of samples.
  • the PCR reagents comprise a DNA polymerase
  • deoxyribonucleotide triphosphate dNTP
  • a reaction buffer a reaction buffer
  • at least one primer a primer
  • the polymerase chain reaction reagent mixture can be present in a dried form, such as a "Ready-to-GoTM" (RTG) format.
  • RTG Ready-to-GoTM
  • the advantage of dried or lyophilised formulations of the polymerase chain reaction reagents is that they can be easily solublised by the addition of water, thus saving operator time and facilitating operator usage.
  • the dried reagent mixture can be pre-dispensed into the reaction vessel, such as the well of a multi-well plate.
  • RTG mixture examples include "Illustra Ready-to-Go RT-PCR beads” available from GE Healthcare (product code: 27- 9266-01 Illustra Ready-To-Go RT-PCR Beads). These freeze-dried beads that include the reagents necessary for one-step reverse transcription-PCR, can be pre-dispensed into a reaction vessel, such as the well of a multi-well plate, as a single dose ready for use.
  • the preformulated, predispensed, ambient-temperature-stable beads thus ensure greater reproducibility between reactions, minimize pipetting steps, and reduce the potential for pipetting errors and contamination.
  • a method for amplification of nucleic acid using the polymerase chain reaction comprising the steps: i) contacting the solid matrix as hereinbefore described with a cellular sample
  • the reaction vessel is a well in a multi-well plate.
  • Multi-well plates are available in a variety of formats, including 6, 12, 24, 96, 384 wells (e.g.
  • a method of detecting the amplified nucleic acid using a detection system comprising the steps: i) amplifying the nucleic acid using the method of the second or third aspect hereinbefore described, and
  • the detection system can be a PCR imaging system.
  • a method of quantifying amplified nucleic acid using a detection system comprising the steps:
  • the sample is a cellular sample.
  • the cellular sample may originate from a mammal, bird, fish or plant or a cell culture thereof.
  • the cellular sample is mammalian in origin, most preferably human in origin.
  • the cellular sample is selected from a group consisting of viral, bacterial, tissue culture, blood and buccal cells.
  • the sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g.
  • the nucleic acid is immobilised on the solid support for at least 24 hours.
  • the nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 7 days, for at least 30 days, for at least 90 days, for at least 180 days, for at least one year, and for at least 10 years.
  • the nucleic acid may be stored in a dried form which is suitable for subsequent analysis.
  • samples are stored at temperatures from -200°C to 40°C.
  • stored samples may be optionally stored in dry or desiccated conditions or under inert atmospheres.
  • the portion is transferred to the reaction vessel by punching or cutting a disc from the solid support.
  • Punching the portion or disc from the solid support can be effected by use of a punch, such as a Harris Micro Punch (Whatman Inc.; Sigma Aldrich)
  • the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool and a forensics tool.
  • kits for storage and amplification of nucleic acid comprising a solid matrix as hereinbefore described and instructions for use thereof.
  • the solid matrix of the kit further comprises a lyophilised or impregnated or printed Ready to Go PCR reagent for one step nucleic acid amplification wherein the Ready to Go PCR beads comprise of a DNA polymerase,
  • dNTP deoxyribonucleotide triphosphate
  • Figure 1 A shows a configuration of a solid matrix (10) whereby the lysis reagent (20), sequestrant (40) and PCR reagents (50) are physically separated by a spacer (30).
  • Figure IB shows a configuration of a solid matrix (60) whereby the lysis reagent (70), sequestrant (90) and PCR reagents (100) are physically separated by a spacer (80).
  • Figure 2 shows the results from PCR amplification of unwashed blood-spotted FTA with or without liquid a-cyclodextrin treatment.
  • Figure 3 shows the results from PCR amplification of unwashed blood-spotted FTA combined with a a-cyclodextrin spotted sample paper.
  • FTA and 31-ETF papers for storing nucleic acid were obtained from GE Healthcare UK Limited;
  • Genomic DNA (Promega product code G152A);
  • the standard well of the 96 well PCR plate was loaded with 5 ⁇ 1 of the 1Kb DNA ladder with 1 ⁇ of 6X loading buffer.
  • control lane was prepared using lng genomic DNA in the presence of 1.25% liquid a-cyclodextrin.
  • Samples were combined with forward and reverse ⁇ -globin primer (lOpmoles/ ⁇ ), a-cyclodextrin (10%) and sterile water for a final volume of 25 ⁇ .
  • the 25 ⁇ sample mix was added to each well of a 96 well PCR plate containing 1 Pure Taq Ready-To-Go PCR bead prior to amplification for a final volume of 25 ⁇ .
  • the thermal cycling conditions were: 95°C for 5min, 95°C for 30sec, 55/65°C for lmin, 72°C for 2min followed by 35 cycles of: 95°C for 30sec, 55/65°C for lmin, 72°C 2min, followed by 72°C for lOmins.
  • visualisation of PCR products was achieved using agarose gel electrophoresis (I X TAE buffer, 1% agarose gel). The results are presented graphically in Figure 2 and 3.
  • Figure 2 shows PCR results of unwashed blood-spotted FTA with or without liquid a-cyclodextrin treatment: Lane 1-3: purified genomic DNA with liquid
  • Lane 4-6 punch spotted with whole blood (1.2mm) with liquid
  • Lane 7-8 no DNA template with cyclodextrin
  • Lane 9-11 purified genomic DNA without cyclodextrin
  • Lane 12-14 punch spotted with whole blood (1.2mm) without cyclodextrin
  • Lane 15-16 no DNA template or cyclodextrin.
  • Figure 2 presents DNA levels obtained from dried blood spots treated with or without cyclodextrin using endpoint PCR. As can be seen, high yields of nucleic acid were obtained from liquid cyclodextrin treated blood spotted FTA samples but PCR was inhibited in the absence of cyclodextrin on blood spotted FTA samples.
  • Table 1 Concentration of reagents in lanes 1 to 16 of Figure 2.
  • FTA ⁇ aliquots of normal human blood were applied to FTA and were allowed to dry and 10% liquid a-cyclodextrin was applied to 31-ETF (final concentration of 1.2%). Or FTA was pre-spotted with 10% cyclodextrin and dried prior to the addition of blood. Punches (1.2mm diameter of FTA and 3mm diameter of 31-ETF) were extracted from each paper type ( ⁇ normal human blood) using the Harris Uni-core punch. Both punches were added to the PCR well along with the reaction mix.
  • Figure 3 shows PCR results of unwashed blood-spotted FTA with or without a- cyclodextrin treated 31-ETF: Lane 1-3: blood spotted FTA (1.2mm) with cyclodextrin spotted 31-ETF (3mm); Lane 4-6:blood spotted FTA (1.2mm diameter punch) with cyclodextrin spotted 3 l-ETF(3mm); Lane 7-9: blood spotted and cyclodextrin spotted FTA (1.2mm diameter punch); Lane 10-12: blood spotted and cyclodextrin spotted FTA (3mm diameter punch); Lane 13-15: blood spotted FTA (1.2mm diameter) with liquid cyclodextrin added; Lane 16-17 no DNA template; Lane 18-19: purified genomic DNA with 1.25%) liquid cyclodextrin.
  • Figure 3 presents DNA levels obtained using endpoint PCR from blood spotted

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Abstract

La présente invention concerne des matrices, des procédés et des trousses pouvant être utilisés pour amplifier de l'acide nucléique, et qui présentent l'avantage de réduire le temps de l'utilisateur ainsi que le risque de contamination de l'utilisateur. Les réactifs de PCR sont liés à une matrice solide afin de faciliter le traitement et l'amplification d'échantillons d'ADN.
PCT/EP2013/068947 2012-09-13 2013-09-12 Matrice solide pour l'amplification d'acide nucléique en une étape WO2014041093A1 (fr)

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GB1502256.9A GB2519465B (en) 2012-09-13 2013-09-12 Solid matrix for one step nucleic acid amplification

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GBGB1216387.9A GB201216387D0 (en) 2012-09-13 2012-09-13 Solid matrix for one step nucleic acid amplification
GB1216387.9 2012-09-13
US13/799,174 2013-03-13
US13/799,174 US9260711B2 (en) 2012-09-13 2013-03-13 Solid matrix for one step nucleic acid amplification

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014143714A2 (fr) * 2013-03-15 2014-09-18 Ge Healthcare Uk Limited Procédés d'amplification d'acides nucléiques d'échantillons non élués en une étape
EP3421991A4 (fr) * 2016-02-23 2019-11-06 Noul Co., Ltd. Timbre de réaction en chaîne par polymérase, et procédé et appareil de diagnostic l'utilisant
US11360005B2 (en) 2016-02-23 2022-06-14 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5496562A (en) * 1988-10-05 1996-03-05 Flinders Technologies Pty Ltd Solid medium and method for DNA storage
WO1999038962A2 (fr) * 1998-02-02 1999-08-05 Gentra Systems, Inc. Compositions et procedes d'utilisation d'une matrice lytique pour isoler un adn
WO2000004195A1 (fr) * 1998-07-16 2000-01-27 Invitrogen Corporation Archivage de vecteurs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5496562A (en) * 1988-10-05 1996-03-05 Flinders Technologies Pty Ltd Solid medium and method for DNA storage
WO1999038962A2 (fr) * 1998-02-02 1999-08-05 Gentra Systems, Inc. Compositions et procedes d'utilisation d'une matrice lytique pour isoler un adn
WO2000004195A1 (fr) * 1998-07-16 2000-01-27 Invitrogen Corporation Archivage de vecteurs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CARLSTEDT JONAS ET AL: "Release of DNA from surfactant complexes induced by 2-hydroxypropyl-beta-cyclodextrin.", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 1 MAR 2010, vol. 46, no. 2, 1 March 2010 (2010-03-01), pages 153 - 158, XP002714920, ISSN: 1879-0003 *
FAN ET AL.: "Supramolecular assembly of cyclodextrin-based nanospheres for gene delivery", JOURNAL OF CONTROLLED RELEASE, vol. 152, 2011, XP002714921 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014143714A2 (fr) * 2013-03-15 2014-09-18 Ge Healthcare Uk Limited Procédés d'amplification d'acides nucléiques d'échantillons non élués en une étape
WO2014143714A3 (fr) * 2013-03-15 2014-12-04 Ge Healthcare Uk Limited Procédés d'amplification d'acides nucléiques d'échantillons non élués en une étape
US10053686B2 (en) 2013-03-15 2018-08-21 Ge Healthcare Uk Limited Methods for one step nucleic acid amplification of non-eluted samples
EP3421991A4 (fr) * 2016-02-23 2019-11-06 Noul Co., Ltd. Timbre de réaction en chaîne par polymérase, et procédé et appareil de diagnostic l'utilisant
US11041842B2 (en) 2016-02-23 2021-06-22 Noul Co., Ltd. Culturing patch, culturing method, culture test method, culture test device, drug test method, and drug test device
US11208685B2 (en) 2016-02-23 2021-12-28 Noul Co., Ltd. Diagnostic method and device performing the same
US11360005B2 (en) 2016-02-23 2022-06-14 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof
US11366043B2 (en) 2016-02-23 2022-06-21 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof
US11385144B2 (en) 2016-02-23 2022-07-12 Noul Co., Ltd. Antibody-providing kit, antibody-containing patch, method and device for immunoassay using the same
US11740162B2 (en) 2016-02-23 2023-08-29 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof
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