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WO1996000301A1 - Reactifs encapsules pour amplification pcr - Google Patents

Reactifs encapsules pour amplification pcr Download PDF

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Publication number
WO1996000301A1
WO1996000301A1 PCT/US1995/007959 US9507959W WO9600301A1 WO 1996000301 A1 WO1996000301 A1 WO 1996000301A1 US 9507959 W US9507959 W US 9507959W WO 9600301 A1 WO9600301 A1 WO 9600301A1
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WO
WIPO (PCT)
Prior art keywords
reagents
matrix
agarose
encapsulated
composition
Prior art date
Application number
PCT/US1995/007959
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English (en)
Inventor
Robert A. Setterquist
G. Kenneth Smith
Original Assignee
The University Of Houston
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The University Of Houston filed Critical The University Of Houston
Priority to AU28710/95A priority Critical patent/AU2871095A/en
Publication of WO1996000301A1 publication Critical patent/WO1996000301A1/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
    • C12Q1/6844—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

  • This invention relates to reagent compositions for enzymatic reactions, and more particularly, to encapsulated reagents for active release at a specified reaction temperature.
  • the invention includes enzymes or other required reagents encapsulated within a matrix and thereby isolated from a reaction mixture, which matrix dissociates at a desired temperature, releasing reactants to initiate a desired reaction.
  • the polymerase chain reaction is a chemical method for greatly increasing or amplifying the concentration of a specific nucleic acid sequence relative to that of other nucleic acids in the reaction mixture.
  • a typical reaction includes the following reagents: (1) a target nucleic acid sequence; (2) at least two oligonucleotide primers complementary to the ends of the target sequence on opposite strands and oriented so that their 3'-OH ends point toward one another along the intervening sequence; (3) a polymerase enzyme; (4) mononucleotides; and (5) divalent metal ion (Mg 2+ ) .
  • the PCR amplification reaction is generally carried out in a thermal cycler which provides repeating temperature cycles of (1) heating to cause strand separation (90-99°C) ; (2) cooling to permit primer-template annealing (30-60°C) ; and (3) warming to permit optimal polymerase activity and extension of the primers (70-75°C) .
  • the optimal temperature for each phase in the ther ocycling will vary with the characteristics of each specific target and set of primers.
  • This cycle is repeated approximately 10-50 times to amplify the target nucleic acid sequence.
  • the typical gain per cycle is slightly less than the theoretical maximum of 2.OX, such that operation through about 30 cycles can cause amplification approximately up to 5 X 10 8 times.
  • PCR technique Many variations of the PCR technique have recently evolved, enhancing this powerful tool for use in nucleic acid sequencing, diagnostic testing, and therapeutics.
  • inherent problems in the technique can cause problems of fidelity, resulting, for example, in amplification of nonspecific fragments that complicate or obscure results.
  • the unwanted products are primarily due to nonspecific binding of primers under conditions of low stringency and low level activity of the thermostable polymerase during set-up and early portions of the reaction.
  • Those reactions using complex genomic or cDNA templates, degenerate primers, very low copy number targets, large numbers of thermal cycles (e.g., greater than 35 cycles) , or more than one target sequence in the same tube (multiplex PCR) are particularly prone to the generation of a variety of unwanted side reaction products.
  • Hot Start PCR can improve the specificity and yield of PCR by isolating from the reaction an essential component such as the polymerase enzyme or primers until all other reaction components have been heated above the annealing temperature. The missing component is then added to each PCR reaction tube.
  • hot start PCR can improve PCR performance, existing hot start methods are time-consuming, cumbersome, and prone to cross-contamination.
  • the PCR reaction tube containing all reactants except polymerase or primers is heated until a wax film is melted over the reaction components.
  • the tube is cooled to solidify the wax.
  • the missing reagent is then pipetted atop the wax barrier, and the tubes returned to the thermocycler.
  • the PCR temperature is then increased and the wax melts, allowing the missing reagents to mix with other reactants.
  • the wax barrier hot start is inconvenient due to the extra steps required in setting up the PCR reaction, e.g., melting the wax, cooling the wax, adding reagents and restarting the PCR reaction. Both manual and wax-barrier hot starts are associated with an increased risk of cross-contamination of samples because the tubes must be reopened before the amplification reaction is started. In addition, the wax begins to melt at about 50°C, permitting reactants to mix at temperatures well below typical annealing temperatures. Thus, this method does not solve the fidelity problems associated with PCR.
  • Another variation of the wax barrier technique isolates MgCl from the reaction mixture.
  • the wax begins to melt at about 50°C permitting addition of Mg 2+ to the reaction mixture.
  • This method suffers the same drawbacks as conventional hot start PCR, because all reagents are present at typical annealing temperatures.
  • trace amounts of Mg 2+ are generally present in the reaction mixture and the polymerase retains some activity even in the absence of a divalent metal cation.
  • reagents required for primer extension including nucleic acid polymerase enzyme, can be stably immobilized within a hydrophilic matrix, which matrix is formed of a substance which dissociates at a specified temperature releasing active reagents.
  • polymerase enzyme and other PCR reagents can be encapsulated or entrapped in a matrix such as agarose which dissociates at a given temperature, releasing the reagents.
  • nucleic acid polymerase primers.
  • reagent beads are coated with a barrier film such as paraffin wax to prevent desiccation of the agarose and to provide a condensation barrier, for example during PCR reactions.
  • the reaction beads are added, for example, to a PCR reaction tube containing a sample.
  • the reaction mixture is heated to reach the desired denaturation temperature. If present, a wax coating melts from the reaction bead as the tube is heated at approximately 50-55°C.
  • the agarose matrix does not dissociate to release its entrapped reagents until a predetermined temperature is reached, determined by the melting point of the matrix. In general, the matrix will dissociate between approximately 60°-95°C, depending upon the type and concentration of the matrix used.
  • the encapsulated reagent composition thus provides a true hot start reaction for PCR or other enzymatic reactions.
  • the polymerase enzyme or other reagents are mixed with a melted hydrophilic matrix such as agarose and the mixture is coated onto a reaction surface such as the wells of a microtiter plate or walls of reactions tubes.
  • the coated plates or reaction tubes may be used immediately or stored, preferably sealed from air or moisture for later use. Sample is added to the coated reaction surface, and the thermal cycle begun.
  • the matrix dissociates releasing its entrapped reagents for a true hot start reaction.
  • the encapsulated reagents of the present invention are generally packaged or coated to prevent desiccation and contamination, and are stable at room temperature for long periods of time.
  • compositions can be formulated with specific primers or reagents to provide a simple and reproducible method for one-step PCR reactions.
  • a simple reaction reagent can be formulated as a kit containing encapsulated control and test reagents, initiating PCR with the addition of the encapsulated reagents to a test sample.
  • the reagent composition of this invention can provide pre- aliquoted reagents for reproducible, error-free, and true hot start PCR. Multiple analysis can be accomplished, for example on a microtiter test plate, with specified wells containing varied control and test reagents.
  • Figure 1 is a photograph of an ethidiu bromide- stained agarose gel showing the MSP gene of C. elegans amplified by the reactions described in Example 1.
  • Lane 1 control, agarose bead, no wax, oil atop;
  • Lane 2 control, wax-coated agarose bead;
  • Lane 3 Test-1, wax-coated agarose-encapsulated Tag DNA polymerase;
  • Lane 4 Test-2, wax-coated agarose-encapsulated Tag DNA polymerase and primers.
  • Figure 2 is a photograph of an ethidium bromide- stained agarose gel showing the MSP gene of C. elegans amplified in the presence and absence of human blood (lanes 1 and 2) and the human beta-globin gene amplified from a human blood sample (lanes 3-5) by the reactions described in Example 2.
  • Lane 1 Control-1, MSP gene, no agarose, no blood
  • Lane 2 Control-2, MSP gene, no agarose, blood
  • Lane 3 Control-3, h3G gene, no agarose, oil atop
  • Lane 4 Test-1, h3G gene, wax-coated agarose- encapsulated Tag DNA polymerase;
  • Lane 5 Test-2, h3G gene, wax-coated, agarose- encapsulated Tag DNA polymerase and primers.
  • Figure 3 is a photograph of an ethidium bromide- stained agarose gel showing MSP gene of C. elegans amplified by the reactions described in Example 4.
  • Lane 1 control, agarose coated onto reaction tube
  • Lane 2 test, agarose-encapsulated Tag DNA polymerase coated onto reaction tube.
  • Figure 4 is a photograph of an ethidium bromide- stained agarose gel showing the MSP gene of C. elegans amplified by the reactions described in Example 5.
  • Lane 1 control, Tag DNA polymerase kept at room temperature 30 days prior to PCR;
  • Lane 2 test, agarose-encapsulated Tag DNA polymerase kept at room temperature 30 days prior to PCR.
  • Figure 5 is a photograph of an ethidium bromide- stained agarose gel showing the MSP gene of C. elegans amplified by the reactions described in Example 6.
  • Lane 1 Low melting point (LMP) agarose-encapsulated polymerase (5.0 units), high copy number (10*);
  • Lane 2 high melting point (HMP) agarose- encapsulated polymerase (5.0 units), high copy number (10*); Lane 3: HMP agarose-encapsulated polymerase (2.5 units) , high copy number (10 4 ) ;
  • Lane 4 LMP agarose-encapsulated polymerase (5.0 units), low copy number (10 3 ) ;
  • Lane 5 HMP agarose-encapsulated polymerase (5.0 units) ; low copy number (10 3 ) ;
  • Lane 6 HMP agarose-encapsulated polymerase (2.5 units), low copy number (10 3 ) .
  • the present invention provides a composition for use in chemical reactions which includes reactants encapsulated in a matrix formulated to dissociate and release its contents at a desired reaction temperature. Upon addition of sample and heating to the appropriate reaction temperature, the matrix dissociates, releasing a premeasured amount of reactants into the reaction mixture.
  • the reaction units may be formed as beads or coated onto a reaction surface such as the inner surface of a reaction tube or microtitre well.
  • reactants are encapsulated in a matrix which dissociates at a desired hot reaction temperature.
  • a desired hot reaction temperature For example, it is highly desirable to isolate certain specific PCR reactants such as polymerase enzyme, oligonucleotide primers, mononucleotides, Mg "1-1' , or combinations thereof from the reaction mixture until the reaction temperature has passed annealing temperature, e.g., approximately T m , which generally is greater than approximately 60°C.
  • Annealing temperature is the temperature above which the oligonucleotide primers and the DNA template melt or dissociate.
  • the annealing temperature will vary with the sequence homology between the template and the primers and the length and GC content of the primer and template.
  • the annealing temperature at which 50% of a primer is annealed to its template (T m ) as well as an optimal annealing temperature for a specific reaction can be calculated, for example as described in Rychlik et al., Nucleic Acids Research. 18:6401-6412, 1990. In general, most PCR reactions utilize annealing temperatures in the range of from about 50°C to about 75°C.
  • PCR primer-template annealing The specificity or fidelity of PCR primer-template annealing is a function of temperature. That is, the higher the temperature, the less likely mispriming will occur. It is therefore desirable in many situations to conduct PCR at high stringency (high temperatures) , e.g., greater than 60°C. Prior to the present invention, no hot start technique save manual reagent addition could exceed typical annealing temperatures, and all are therefore prone to mispriming and subsequent spurious PCR product formation.
  • Hot start PCR reactions have been achieved by isolating polymerase, primers, mononucleotides, or magnesium from other reactants with a barrier film of paraffin wax.
  • the wax melts at approximately 50-55°C, permitting mixing of the previously isolated reactants with the reaction mixture. While this method may improve the quality of the PCR reaction, it does not achieve a true hot start PCR reaction above annealing temperatures and still suffers from some non-specific priming.
  • the polymerase enzyme e.g., DNA polymerase such as Tag DNA Polymerase and/or other reactants into a matrix such as agarose
  • the matrix is preferably hydrophilic, and preferably dissociates at a reaction temperature greater than approximately 60°C.
  • the most preferred dissociation temperature range is from about 6 °C to about 95°C.
  • a preferred dissociation temperature is about 90°C.
  • Low melting point agarose is commercially available, and melts or dissociates at approximately greater than 60°C.
  • High melting point agarose dissociates at approximately greater than 90°C.
  • Other forms of agarose are commercially available which exhibit melting points between 60°C and 95°C. See, for example, the 1992 Sigma Chemical Co. catalog and the 1994 FMC Bio Products Catalog, which show types of agarose having varied melting temperatures which may be purchased. By varying the type and/or concentration of a particular type of agarose, a variety of specific remelting temperatures can be achieved, e.g., compositions having dissociation temperatures between approximately 60°C and 95°C can be formulated.
  • a type of agarose have a melting point in the generally desired remelting temperature range is selected. For example, if the desired remelting temperature is approximately 92°C, high melting point agarose is selected (MP « 94°C) . Agarose solutions of various concentrations (e.g. 0.1-1.0%) are prepared in aqueous solution, e.g. , water, heated to melting and cooled to gelling point in a reaction tube. The agarose reaction tubes are then reheated and the remelting points observed. In this way, an appropriate agarose and its concentration, for example that which remelted at 92°C can be selected.
  • aqueous solution e.g. , water
  • nucleic acid polymerization includes any reaction in which a nucleic acid sequence is extended by the addition of mononucleotides. Nucleic acid polymerization includes, for example, PCR, nucleic acid sequencing, reverse transcriptase polymerization and nick translation. See, for example, Focus 16:38-57 (1994).
  • the composition of the present invention includes one or more reagents required to initiate nucleic acid polymerization, the reagent(s) encapsulated within a matrix which dissociates at a predetermined temperature.
  • the encapsulated reagents include one or more of nucleic acid polymerase, primers, mononucleotides, and divalent metal cations.
  • the composition includes nucleic acid polymerase and/or primers.
  • PCR reagents including polymerase, primers, mononucleotides, and divalent metal cations, remain stable at room temperature when encapsulated within an agarose matrix.
  • the storage time of the encapsulated reagents may be extended by coating the surface of the matrix with a barrier to avoid desiccation of the matrix, e.g., with a thin coating of paraffin or by storing in an evacuated or sealed container.
  • DNA polymerase encapsulated in agarose beads and dipped in paraffin remains stable at room temperature for at least several months. Variations in the above-described invention will be obvious to those of skill in the art.
  • the encapsulating matrix may be formed of a variety of materials, polymers, and the like, provided the matrix dissociates at the desired reaction temperature and is compatible with the desired reaction, that is, does not interfere with the reaction sequence or its products.
  • the reaction is carried out in the presence of the matrix over a range of matrix concentrations. The reaction is monitored for interference as compared with a matrix-free control.
  • any number of reagents may be encapsulated within the matrix, providing a unique method of quality control and reproducability of reaction conditions. Reagent contamination, inactivation and waste are greatly reduced by the single-reaction unit provided by the instant composition.
  • the matrix may include polymerase and/or primers, and optionally all other necessary reactants such as buffers, salts, mononucleotides, and the like. In this way, a user simply adds the encapsulated reagent unit to a reaction tube containing sample. No further additions are required.
  • the one or more reactants are mixed with molten matrix, and the mixture is permitted to gel, encapsulating the reagents.
  • the reactants may include solid, solvated, or aqueous solutions or dispersions of the compounds.
  • aqueous solutions of the reactants are mixed with the molten matrix, e.g., molten agarose.
  • the encapsulated reagents include enzymes, such as polymerase enzyme for PCR reactions.
  • Polymerase enzymes are commercially available, such as Tag DNA polymerase. Vent DNA polymerase, Tth DNA polymerase, and the like (New England Biolabs) .
  • a lOO ⁇ l PCR uses approximately 1-5 units of polymerase, typically available in about 1-2.5 ⁇ l of the commercial enzyme.
  • enzyme is added to molten matrix to yield a desired final reaction concentration.
  • an amount of Tag DNA polymerase (1-10 units) can be added to 5-9 ⁇ l molten agarose and formed into a lO ⁇ l bead.
  • agarose-Tag polymerase compositions successful PCR with as little as 1.25 units of polymerase, even at low template copy number has been achieved.
  • Oligonucleotide primers may also be encapsulated in the matrix. Generally, the amount of primer added to matrix is sufficient to yield approximately 0.1-l ⁇ M of each primer in the final reaction volume. Primers are typically at least 15 base pairs in length, and generally range from 30-60 base pairs. Primers may also include restriction enzyme sites and may be labeled, e.g., radio, fluorescent or biotin-labeled.
  • Mononucleotides are essential to the elongation of primers in PCR and other polymerization methods.
  • Monomers may be encapsulated in the matrix, generally in a concentration range of about 50 to about 200 ⁇ M for each monomer.
  • Useful monomers are deoxyadensosine, guanosine, cytosine and thy ine triphosphates, (dNTPs) and analogs thereof.
  • Monomers may be labeled or modified as known to those of skill in the art.
  • the encapsulated reagent unit may be adapted by one of skill in the art for particular reactions.
  • a multi-stage unit can be used.
  • An initial matrix e.g., of high melting point agarose (>90°C) is used to encapsulate a first specific primer, and/or other reactants and is formed into a bead.
  • This bead is then coated with a second matrix, e.g., of low melting point agarose (>60°C) encapsulating a different (second) set of primers, and optionally DNA polymerase and other PCR reactants.
  • the multi-layered bead is preferably coated with paraffin wax.
  • the multi-layered bead In its use in the PCR reaction, the multi-layered bead is first run through approximately 10 "cold" cycles with a maximum temperature of less than 90°C causing reaction with the second set of primers. Continuing cycles are then run at approximately >94°C to invoke reaction with the first specific set of primers. Variations in these techniques will be readily apparent to those of skill in the art.
  • Control agarose without any entrapped reagents was also used.
  • the agarose solutions were dropped onto an ice cooled parafilm sheet to form beads .
  • the beads were then dipped 3 times into melted paraffin wax.
  • PCR was carried out using as sample template the plasmid pCEOl.
  • pCEOl was constructed by digesting the plasmid p3L4 (Ward et al . , Journal of Molecular
  • brackets indicate reagents encapsulated within agarose bead
  • the first control reaction was run according to traditional PCR procedure, with mineral oil placed atop the reaction mixture to avoid condensation.
  • a wax-coated agarose bead containing no PCR reactants was added to the reaction mixture.
  • agarose beads having incorporated polymerase or polymerase and primers as discussed above were used.
  • the reaction tubes were capped and placed in a Perkin Elmer Cetus DNA Thermal Cycler Model No. 480 (Perkin Elmer, Norwalk, CT) .
  • the PCR conditions included approximately three minutes heating to reach 94°C, followed by repeating cycles of one minute at 94°C; thirty seconds at 58°C; and thirty seconds at 72°C for thirty cycles.
  • beta-globin Amplification of beta-globin from human blood
  • Whole blood was collected into a sample tube after pricking a human finger with a sterile lancet.
  • One microliter of whole blood was used in each reaction mixture as sample from which beta-globin was to be amplified.
  • the primers PI and P2 as described in Bauer, et al., JAMA 265:472-477, 1992 were used to amplify the 293 base pair human beta-globin gene.
  • the primer sequences were:
  • the MSP gene was amplified from C. elegans DNA as described for Example 1 in the presence or absence of 1 ⁇ l human blood.
  • the reaction mixtures were set up as follows :
  • brackets indicate reagents encapsulated within agarose bead
  • the reaction tubes were placed in a thermal cycler and PCR initiated and carried through 30 cycles as described for Example 1.
  • the results of the PCR are shown in Figure 2 and demonstrate improved reaction product when the human beta-globin gene is amplified from a blood sample by the hot start agarose bead methods over conventional PCR.
  • the control, conventional PCR shows very little production of the desired 293 base pair product and the presence of unwanted bands.
  • both lanes showing reaction product produced from the inventive agarose bead hot start reactions clearly showed the desired 293 base pair product with little or no unwanted bands.
  • Agarose beads with varied melting points Agarose beads formed of varying concentrations of high melting point agarose were evaluated for the temperature at which they dissociated and released their encapsulated contents. Solutions of 0.25%, 0.5% and 1.0% high melting point agarose were prepared, melted, and mixed with bromophenol blue dye, and formed into 10 ⁇ l beads as described above. The beads were dipped three times in paraffin and placed in lOO ⁇ of water. The tubes were placed in a thermocycler and slowly heated to controlled temperatures. The dissociation temperature was identified by release of dye from the bead into the surrounding solution.
  • Low melting point agarose (0.8%) was prepared as described for Example 1 with and without Tag DNA Polymerase.
  • the molten agarose-reagent solution was coated onto the reaction surface of 500 ⁇ l reaction tubes by dripping 10 ⁇ l of the agarose-reagent solution into a tube and swirling to coat the bottom and the lower portion of the side walls of the tube.
  • the tubes were cooled at room temperature for approximately 10 minutes to allow the agarose to gel.
  • the reaction tubes were set-up by adding the appropriate reagents to the test tubes, and layering approximately 40 ⁇ l mineral oil atop each mixture.
  • brackets indicate reagents within agarose
  • PCR was carried out and results analyzed as described for Example 1. As shown in Figure 3, both control and test lanes show the target, 380 base pair MSP band. Incorporation and immobilization of PCR reagents onto a reaction surface provided an efficient hot start PCR product.
  • PCR was performed as described for Example 1, the only difference between the samples being the presence of Tag DNA polymerase within or outside the agarose bead. Results are shown in Figure 4, and demonstrate greater product yield in the reaction having encapsulated polymerase (lane 2) than the reaction using non- encapsulated polymerase. Further, the data indicate the encapsulated polymerase may be stored at room temperature for long periods of time without loss of activity.
  • EXAMPLE 6 COMPARISON OF HOT START TEMPERATURES Agarose beads containing Tag DNA polymerase were prepared as described for Example 1 either low melting point or high melting point agarose. The remelting temperatures for these agarose preparations were determined to be 64°C and 92°C, respectively, by the method described for Example 3. The higher-melting agarose beads contained either 2.5 units or 5.0 units of Tag DNA Polymerase, the lower-melting point agarose beads contained 2.5 units.
  • PCR was carried out as described for Example 1 using either high copy number (10*) target or low copy number (10 3 ) target, through an initial three minutes at 94°C, followed by 35 cycles of one minute at 94°C, 30 seconds at 50°C; and 30 seconds at 72°C.
  • the amplified product was analyzed as described for Example 1. The results, shown in Figure 5, show amplification of the desired 380 bp product in all samples including high copy number (lanes 1-3) and low copy number (lanes 4-6) .

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Abstract

Compositions et procédés servant à réaliser des réactions enzymatiques à déclenchement à chaud, notamment l'amplification PCR, et permettant d'obtenir des réactifs essentiels encapsulés dans une matrice qui se dissocie lorsqu'une température prédéterminée est atteinte.
PCT/US1995/007959 1994-06-24 1995-06-23 Reactifs encapsules pour amplification pcr WO1996000301A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU28710/95A AU2871095A (en) 1994-06-24 1995-06-23 Encapsulated pcr reagents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26636294A 1994-06-24 1994-06-24
US08/266,362 1994-06-24

Publications (1)

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WO1996000301A1 true WO1996000301A1 (fr) 1996-01-04

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WO2001007581A1 (fr) * 1999-04-23 2001-02-01 Nexttec Gmbh Procede destine a encapsuler des macromolecules et/ou des particules et utilisation de ce procede
KR20020084960A (ko) * 2001-05-03 2002-11-16 김홍찬 차량용 발열시트의 열선구조
WO2003012066A3 (fr) * 2001-08-02 2003-12-04 Wayne M Barnes Procede de demarrage a chaud utilisant un precipite de magnesium pour manipulation moleculaire d'acides nucleiques
WO2003078659A3 (fr) * 2002-03-20 2003-12-24 Innovativebio Biz Microcapsules encapsulant un melange de reaction d'amplification de l'acide nucleique et utilisation de celles-ci en tant que compartiments de reaction pour des reactions paralleles
US6942964B1 (en) 1999-07-09 2005-09-13 Sigma-Aldrich Co. Tracer reagents that enhance reaction-product analysis
WO2005106023A1 (fr) * 2004-04-30 2005-11-10 Siemens Aktiengesellschaft Procede et dispositif d'amplification d'adn par amplification en chaine par polymerase au moyen de reactifs secs
WO2006036845A1 (fr) * 2004-09-24 2006-04-06 Cepheid Billes de reactif specifique de cible universelles pour amplification d'acide nucleique
WO2006036848A3 (fr) * 2004-09-24 2009-04-02 Cepheid Systeme de reactif a plusieurs billes pour analyses basees sur des proteines avec matrices optimisees
US20090186357A1 (en) * 2007-12-10 2009-07-23 The Trustees Of The University Of Pennsylvania Integrated PCR Reactor for Cell Lysis, Nucleic Acid Isolation and Purification, and Nucleic Acid Amplication Related Applications
JP2011160728A (ja) * 2010-02-10 2011-08-25 Sony Corp 核酸増幅反応用マイクロチップ及びその製造方法
US8088576B2 (en) * 2004-04-30 2012-01-03 Siemens Aktiengesellschaft Method and assembly for DNA isolation with dry reagents
US8187557B2 (en) 2006-07-13 2012-05-29 Cepheid Reagent reservoir system for analytical instruments
EP2602331A1 (fr) * 2011-12-09 2013-06-12 Qiagen GmbH Réactif de diagnostic intégré dans la cire en tant que référence interne pour la préparation ou la détection d'acides nucléiques
US8911938B2 (en) * 2007-12-10 2014-12-16 The Trustees Of The University Of Pennsylvania Reaction chamber having pre-stored reagents
US9194226B2 (en) 2013-08-01 2015-11-24 Tyler W. Blair Oil and gas fracture liquid tracing using DNA
US20200239944A1 (en) * 2017-07-04 2020-07-30 Korea Institute Of Science And Technology Particle with ucst material applied thereto, and nucleic acid amplification method using same
CN114703259A (zh) * 2022-04-14 2022-07-05 苏州天隆生物科技有限公司 一种核酸检测试管的分隔物、包含其的检测试管与扩增方法
DE102024203913B3 (de) * 2024-04-25 2025-04-17 Robert Bosch Gesellschaft mit beschränkter Haftung Beschichtung für ein Kavitätenarray für eine mikrofluidische Vorrichtung in der Point-of-Care-Diagnostik

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WO1991012342A1 (fr) * 1990-02-16 1991-08-22 F. Hoffmann-La Roche Ag Ameliorations apportees a la specificite et a l'applicabilite de la reaction en chaine de polymerases
EP0572057A1 (fr) * 1992-05-11 1993-12-01 Johnson & Johnson Clinical Diagnostics, Inc. Composition et kit de réactifs de PCR et procédés pour amplification et détection avec amplification non-spécifique d'acides nucléiques réduite

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