CN101516337A - Preparation of glassified biological reagents - Google Patents
Preparation of glassified biological reagents Download PDFInfo
- Publication number
- CN101516337A CN101516337A CNA2007800345077A CN200780034507A CN101516337A CN 101516337 A CN101516337 A CN 101516337A CN A2007800345077 A CNA2007800345077 A CN A2007800345077A CN 200780034507 A CN200780034507 A CN 200780034507A CN 101516337 A CN101516337 A CN 101516337A
- Authority
- CN
- China
- Prior art keywords
- reagent
- exsiccant
- mixture
- preparation
- pcr
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention related to a method of making a dried reagent preparation, comprising the steps of: providing an aqueous solution of at least one buffered biological reagent; mixing a glass forming filler material with the buffered reagent solution to form a mixture wherein the concentration of the filler material is sufficient to facilitate formation of a glassy, porous composition; dispensing the mixture in the form of substantially uniform droplets into wells of a multi-well container, wherein a single droplet is dispensed into each well; drying the droplets in the container to form the reagent preparation. The reagent preparation is water soluble and has a Tg sufficient for room temperature stability.
Description
Cross
The priority of 60/887,364 (its disclosure is incorporated this paper into as a reference in full with it) that the application requires that JIUYUE in 2006 submitted on the 18th U.S. Provisional Patent Application is submitted to number on January 31st, 60/845,307 and 2007.
Invention field
The present invention relates to biologic material and reagent with glassy, porous state long term store.Especially, it relates to the use porous plate and prepares the method for these materials and reagent and its storage.
Background of invention
The minority biologically active material is enough stable, thus they can at room temperature separate, purification and in solution, storing then.Usually, biological reagent is stored in the glycerite that remains on 4 ℃ ,-20 ℃ or-70 ℃.Can they are in bulk (in bulk) store, then before use with other agent combination.
Be used for the facility of biological sample and the reagent of effectively testing in preparation, often importantly obtain even, the pre-exsiccant chemical admixture of estimating (discreet amount).Being used for the carrier of stabilate reagent or a type of implant is the packing material that forms glass.Biological reagent solution is incorporated into the packing material (described material is water solublity or water-swellable material) that forms glass.Then their dryings are fixed the glassy compositions of learning reagent with stabilate to produce.About being used for the packing material of formation glass that stabilate learns reagent referring to US 5,098,893, US5,200,399 and US 5,240,843.
Carbohydrate for example glucose, sucrose, maltose or maltotriose is the material group of important formation glass.Can use other polyols for example carbohydrate derivates such as Sorbitol and through the carbohydrate of chemical modification.The material of another kind of important formation glass is synthetic polymer, as polyvinylpyrrolidone, polyacrylamide or polymine.
The other example that forms the material of glass comprise the saccharide copolymer for example by GEHealthcare at registered trade mark FICOLL
TMThose saccharide copolymers of following sale.FICOLL
TMHave 5,000 to 1,000,000 molecular weight and comprise the sucrose residue (US 3,300,474) that is connected with bifunctional group by the ether bridged bond.This kind group can be 2,3 or more a plurality of carbon atom but be no more than the alkylidene of 10 carbon atoms usually.Bifunctional group is used for saccharide residue is linked together.This kind polymer can for example prepare by the reaction of sugar with halohydrin or bis-epoxy (bis-epoxy) chemical compound.
In the glassy matrices of carbohydrate polymer can be through stable biologic material by lyophilization (people US 5,593,824 such as Treml; Franks and Hatley US 5,098,893) or prepare by vacuum drying people US 5,565,318 such as () Walker.These water-soluble reagents are advantageously used in complicated molecular biology and use.This method specifically is used for the reagent system be made up of enzyme, nucleotide and other components that the aliquot of using with single is distributed.Vitrophyric reconstruction send be used for using (comprising DNA cloning and dna sequencing) through buffered enzyme.
On market, there are many exsiccant molecular biology products at present.Yet some in these products are by suitable trouble and comprise that the method for a large amount of manual labours prepares.Other products need freezing when dry.Existence is to being used to produce the needs of improving one's methods of the exsiccant reagent of ambient temperature.
Summary of the invention
In a first aspect of the present invention, provide the method that produces exsiccant reagent formulation.The method comprising the steps of: at least a aqueous solution through buffered biological reagent is provided; The packing material that forms glass is formed the mixture that the concentration of packing material wherein is enough to promote glassy porous compositions formation with mixing through buffered reagent solution; The mixture of the amount of pre-determining is dispensed into the hole of porous container; With the mixture drying in the container to form exsiccant reagent formulation; Wherein said reagent formulation is water miscible and has for room temperature stability is enough Tg.Mixture is the solution of homogeneous preferably.
In one embodiment of the invention, exsiccant reagent formulation is collected into the room temperature storage to be used to prolong in the reagent bottle.
In another embodiment of the invention, exsiccant reagent is stored in the storage to prolong in the porous container, with the top of band sealed container.Randomly, band is heat activated.
According to porous container of the present invention can be silicon dioxide mold (silica mould) or polystyrene board.When porous container was polystyrene board, the present invention placed polystyrene board on the metal moulds before also being included in lyophilizing, thereby the outer wall of each polystyrene plate hole closely contacts to carry out effectively heat passage with the hole of described metal moulds.We find the exsiccant reagent that this has improved drying means and has produced the integrity with raising.
In another aspect of the present invention, the invention provides exsiccant biological reagent compositions according to method for preparing.
Reagent composition prepared in accordance with the present invention is water miscible and has for holding room temperature stability is enough T
gPreferably, reagent composition has structural intergrity.
The biological reagent compositions can be shorter than 1 minute, is dissolved in fully in preferred 30 seconds in the 25 μ l aqueous solutions.Reagent composition preferably has and is lower than 10% water capacity.
Reagent composition can have at least a when under the room temperature in aqueous solution unsettled reagent during individualism.Reagent composition also can comprise multiple can react each other or can nonreactive reagent when at room temperature existing in aqueous solution.
Therefore, objects and advantages of the present invention provide exsiccant biological reagent compositions and produce described method for compositions.
According to following description, these and other purposes of the present invention and advantage will become apparent.Yet this description only is a preferred embodiment.Therefore, should rely on claim and estimate gamut of the present invention.
Accompanying drawing is described
Fig. 1 shows the method workflow that is used for preparing according to embodiment of the present invention the method for exsiccant reagent formulation.
Fig. 2 shows, from the upper left corner: the 384 hole polystyrene plates that are used for preparing according to embodiment of the present invention the method for exsiccant reagent formulation; The upper right corner: show that having 1,000 ten thousand is copied to the standard curve of the not commensurability λ DNA of 1000 initial concentrations that copy as template, comprises no template contrast; The lower left corner: from the exsiccant reagent formulation cake/cubic block (cube) the plastic bottle of being stored in of 384 hole polystyrene plates preparations; The lower right corner: show that exsiccant reagent formulation is successfully used to the amplification figure of the real-time qPCR amplified reaction of λ DNA.
Fig. 3 shows, from the upper left corner: be used for preparing according to embodiment of the present invention 96 hole silicon dioxide molds of the method for exsiccant reagent formulation; The upper right corner: show not commensurability λ DNA with 1,000 ten thousand initial concentrations that are copied to 1000 copies standard curve, comprise the contrast of no template as template; The lower left corner: from the exsiccant reagent tablet the plastic bottle of being stored in of 96 hole silicon dioxide molds preparation; The lower right corner: show that exsiccant reagent formulation is successfully used to the amplification figure of the real-time qPCR amplified reaction of λ DNA.
Fig. 4 shows that the 96 hole PCR plates that comprise the PCR preparation advance into metal rack (metal holder) in lyophilizing.
Fig. 5 shows the PCR reagent of multi-form ambient-temp-stable according to embodiment preparation of the present invention.The upper left corner: the exsiccant PCR mixture in the bottle.The upper right corner: the exsiccant PCR mixture in 96 orifice plates.The lower left corner: the exsiccant PCR mixture in 384 orifice plates.The lower right corner: the exsiccant PCR mixture in the 96 hole perforated plates (perforated plate).
Fig. 6 shows the stability according to the exsiccant PCR reagent of embodiment of the present invention preparation.Exsiccant PCR mixture is used for the qPCR of λ DNA.Use the exsiccant cake (upper right side) of current rules preparation and (3) puRe taq Ready-To-Go for (1) ' wet ' preparation pearl mixture (upper left side), (2) from GEHealthcare
TM(RTG) PCR pearl (lower left) is noticed similar performance.Lower right: the Ct value of above-mentioned three reactions of carrying out in triplicate and PCR efficient.Ct value and the PCR efficiency value of new model and pure Taq pearl are suitable.
Fig. 7 shows the stability according to the exsiccant PCR reagent of embodiment of the present invention preparation.Exsiccant PCR mixture was stored 8 days down in 40 ℃ and room temperature (RT).Exsiccant reagent is used for the qPCR of λ DNA, has similar performance.The upper left corner: the exsiccant PCR reagent cake of under RT, storing (reagent cake).The upper right corner: in 40 ℃ of exsiccant PCR reagent cakes of storing down.The lower left corner: the puRe Taq pearl (GE Healthcare) that under RT, stores.The lower right corner: in 40 ℃ of puRe Taq pearls that store down.
Fig. 8 shows the Ct value and the PCR efficient of above-mentioned four reactions of carrying out in duplicate.Ct value of current form and pure Taq pearl and PCR efficiency value are suitable under 40 ℃ and room temperature.
Fig. 9 shows the result according to the real-time PCR reactions of one embodiment of the present of invention.The picture left above: use freeze dried reagent to comprise the PCR in real time of TaqMan primer and probe.Lower-left figure: freeze dried reagent contrast (lyophilizing is carried out in the not lyophilizing of TaqMan primer and probe, other reagent as among the embodiment 1).Top right plot: commercial puRe Taq RTG pearl contrast (every other reagent does not carry out lyophilizing).When puRe Taq RTG reaction is used to produce standard curve and other reactions are considered as when unknown, the reaction with template DNA of equivalent drops on (bottom-right graph, " X " expression " the unknown ") on the standard curve.
Figure 10 shows that the Phi29 archaeal dna polymerase is stable with freeze dried form.The whole genome amplification that uses freeze dried reagent or " wetting " mixture to carry out is measured.Even under the situation of using the freeze dried reagent of storing 35 days under 40 ℃, detect intensive amplification.
Figure 11 shows with " wetting " test kit (Roche IVT) of routine and compares, and uses the success of the dna profiling that freeze dried IVT reagent (A, B, C) carries out to transcribe.Ntc: no template contrast.
Detailed Description Of The Invention
Biological reagent
Many biological reagents are fit to store by method of the present invention. Biology examination of the present invention The agent composition is particularly suitable for carrying out many kinds valuably or necessarily to the blood of blood plasma or dilution The routine analyzer that slurry carries out. Routine analyzer usually need to be with blood plasma and one or more reagent balls (reagent sphere) combination, thus in blood plasma, take place can with specific components or the feature of blood plasma Relevant some optics of measurement on detectable variation. Preferably, blood plasma causes experience can pass through Color, the fluorescence, luminous of the change that conventional spectrophotometer, fluorescence photometer, photodetector etc. are measured Deng reaction or other variations. In some cases, can carry out immunoassays and other specificitys knot Close mensuration.
Can be protein and peptide to its biological reagent of using other classifications of the present invention, comprise it Derivative is glycoprotein for example. This kind protein and peptide can be any enzyme, transport protein (transport Protein) (for example hemoglobin, immunoglobulin (Ig), hormone, blooc coagulation factor (blood clotting Factor) and pharmacologically active albumen or peptide).
Can comprise nucleosides, nucleotides to its biological reagent of using another kind of classification of the present invention (for example deoxynucleotide, ribonucleotide and dideoxy nucleotide), dinucleotides, oligonucleotides With also have enzyme cofactor, no matter whether these are nucleotides. Zymolyte also is to use it usually Biological reagent of the present invention.
Being used for can be from natural origin at the stable biological reagent of storage, animal, plant, fungi Or bacterium separates, maybe can be by producing from the cell by fermentation and artificial incubation growth and from its branch From. This kind cell can be or can not be the cell of genetic transformation.
Another development of the present invention is that to store surpassing of reaction system in glass reagent ball a kind of Reagent. This can be used for need to for example measure or diagnostic kit in the material that uses together.
In single glassy preparation store reagents with for final purposes easily form provide They. For example, if measure the combination need substrate or co-factor and enzyme, so can with two kinds or All these three kinds of materials are stored in glassy reagent ball neutralization with the concentration ratio of needs and are ready for Measure.
If the storage plurality of reagents then can mix them in aqueous emulsion, then one Rise and incorporate in the glass. Alternatively, can individually they be incorporated in the glass separately into described separating Glass after be mixed together.
When storing multiple examination as single composition (it can be two kinds of glass that mix) During agent, one or more reagent can be protein, peptide, nucleosides, nucleotides or enzyme cofactor. Reagent may can be simpler kind also. For example, standard test program may need pyruvic acid Salt and NADH are present in together. Both can store separately with acceptable stability. Yet, When placing the aqueous solution together, they begin reaction. If the ratio with needs places glass together In the time of in the glass shape reagent ball, they do not react, and glass can be stored. About reaction, we refer to Any biochemical reaction.
Preferred biological reagent of the present invention provides reagent system to detect, increase, to modify or to survey The enzyme of order nucleic acid and co-factor. This kind enzyme includes but not limited to archaeal dna polymerase (Ke Lienuo for example (Klenow)), T7 archaeal dna polymerase or various heat endurance archaeal dna polymerase Taq DNA for example Polymerase; AMV or mouse reverse transcriptase, T4 dna ligase, T7, T3, SP6RNA gather Synthase, bacteriophage Phi29DNA polymerase and restriction enzyme. Co-factor comprise nucleotides, Oligonucleotides, DNA, RNA, for the essential salt of enzymatic activity (for example magnesium, potassium and sodium) and slow The needed salt of the ability of rushing. Buffer salt provides correct pH scope and has helped stability. Can Some buffers that use comprise Tris pH 7.6-8.3.
Can use the rules of basis embodiment 1 hereinafter to estimate any potential biological reagent.Therefore, make suitable biological reagent stable in the reagent ball, as determined by the functional test among functional test such as the embodiment 1.
Form the packing material of glass
The example that can be used for the packing material of formation glass of the present invention comprises for example FICOLL of carbohydrate
TM, sucrose, glucose, trehalose, melezitose, DEXTRAN
TMAnd mannitol; Protein is BSA, gelatin and collagen for example; And polymer for example PEG and polyvinylpyrrolidone (PVP).The packing material of formation glass is FICOLL preferably
TMPolymer, BSA, sucrose, DEXTRAN
TMOr its combination.Being used for the packing material that most preferably forms glass of the present invention is FICOLL
TMPolymer.
Preparation
Determine the preparation of the high-viscosity mixture of biological reagent, the packing material that forms glass and water by iterative method (iterative process).At first, people determine the whole working concentration that system wishes.Concentration is represented with molarity usually.Each biological reagent can have different prescriptions.The second, these concentration are transformed into weight/dosage basis (basis) (for solid) and volume/dosage basis (for liquid).
The 3rd, the initial value of the percent solids concentration of selection high-viscosity mixture and the volume of mixture of hope.55% solid concentration has shown that performance is good.Be higher than 62% solid concentration, mixture is too sticking so that can not distribute.If Emulsion is wished, be lower than 52% solid concentration, mixture is too rare, and drying is transparent and hard.If the gala agent is wished, and is allowed 10% lower limit.About " solid of % ", we are meant that (solid weight multiply by 100) is divided by (liquid weight+solid weight).
Enter solution if allow to form the material of glass, then mixture is hard with glass with drying.Therefore, the mixture of hope is Emulsion but not solution.About Emulsion, we are meant saturated mixture, thus exist biphase, solid phase and liquid phase.For example, the present invention is the gala agent of the packing material of the formation glass in biological reagent/buffer.Solid existence provides opaque to white to Emulsion.When drying, high viscosity Emulsion still forms glass, passes through with Informal water but can obtain aperture on the surface, and quickens the dissolving of exsiccant reagent formulation.Emulsion should have white.If it is transparent, its most probable is hard with glass with drying, thereby and, will be atresia.About porosity, we are meant that exsiccant preparation reagent comprises the bag that helps the dissolved bubble of described preparation.Preferred porosity allows preparation to dissolve in about 2 minutes or less time.
Another kind of form of the present invention provides and has been characterised in that and is the packing material of the formation glass of gala agent and the mixture of biological reagent/buffer.About this mixture, we are meant the mixture with at least some Emulsion character.Gala agent of the present invention can form by using above-mentioned iterative method to make solid concentration reach about 10% to about 50%.Distribution and dry gala agent form exsiccant reagent formulation then.
The 4th, people's calculating can be used the number from the producible dosage of gram number of the material of every dosage formation glass of second step.
The 5th, the number of using dosage and from the weight/dosage ratio of second step, people determine the weight/volume of other components.At last, use the weight and volume of determining in the 5th step, people calculate the percent solids of whole mixture.If the whole percent solids of mixture outside the scope of hope, then people use another initial value repeat the 3rd to the 6th step until final value in correct scope.
Can use the material of estimating any potential formation glass according to the rules of above-mentioned iterative method.Therefore, the material production of suitable formation glass have acceptable hardness, size, shape, a T
g, porosity, dissolubility and stability reagent formulation.
Porous container
The example that is used to prepare the porous container of exsiccant reagent formulation comprises polystyrene board, silicon dioxide mold etc.Preferably, porous plate is to have the standard size that makes it possible to use the standard thermal cycler that is used for application examples such as PCR and the porous plate of layout.
Usually, porous container comprises two zones, porose area and borders.The border can be any size, shape or thickness, but is preferably formed the porous platform with outside size similar to the outside size of the commercial microtitration plate in standard 96 holes or 384 holes, and its size is taken advantage of length 127.75mm for the about 85.5mm of width.
Usually, plate comprises many cavities that are arranged in the grid.These cavities are called the hole and are used as the reaction vessels of indivedual reactions, or are used for the hold-up vessel of individual samples.The hole will be with the two-dimensional linear arrayed on the porous platform.Yet the hole can for example how much or non-geometric array provide with the array of any kind.The number in hole can be 96 a multiple in these scopes, preferably multiply by 96 integer square.
Hole in the commercial plate has standard usually through design interval.96 orifice plates have 12 row and 8 row, interval 9mm between the center in adjacent hole.384 orifice plates have 24 row and 16 row, interval 4.5mm between the center in adjacent hole.1536 orifice plates have 48 row and 32 row, are spaced apart 2.25mm between the center in adjacent hole.The microtitration plate of half of the above-mentioned form of also use formation.Also obtainable is the pore area that has similar interval between the center in adjacent hole.Have these sizes porous container can for example porous platform changer (translocator) be compatible with reader (calling in this area as them) with robotics (robotics) and instrument.
The material that is used to prepare the porous platform will be generally polymer, because these materials make them be suitable for most of technologies of preparing.Preferably, select known polymer with low fluorescence or other character.The whole bag of tricks in this area can be used for confirming that the polymer of selecting has the character of hope.Polymeric material can promote method of molding by known in the art and exploitation in the future significantly, and for example insert moulding or injection molding carry out the preparation of plate.
The alternative of polymer sheet is the silicon dioxide mold of 96 holes and 384 well format.As shown in the embodiment hereinafter, the plate of 96 holes perforation silicon dioxide molding can be used for distributing and freeze-dried reagent.Advantage herein is should easily take out exsiccant reagent from mold after lyophilizing.
Mix and distribution
Be prepared as follows general preparation (the same dna marker preparation that uses) with embodiment:
Before use, usually employed all reagent are carried out autoclaving or filtration sterilization (filter of preferred 0.25 μ m).Prepare preparation, and it is stored until distribution on ice.For 200ml (being enough to be used in 20,000 single agent formulations) dna marker preparation, the Ficoll 400 of each 15g and Ficoll 70 and 20g melezitose are added about 90ml sterilized water, and on agitating plate, mix until dissolving.
With 50ml 20X concentration of DNA labelling buffer (200mM Tris pH 7.5,200mMMgCl
2, 1M NaCl) add with 10ml10mg/ml BSA solution.100mM ATP, the GTP and the TTP that add each 1ml.In case all reagent exist in solution, just preparation is stored on ice or under refrigerated condition until it and be used for distributing.Tightly before use, add 400Ku Klenow fragment archaeal dna polymerase (least concentration of stock solution should be 100Ku/ml so that glycerol concentration keeps below 1% in whole preparation).Equally tightly before use, the d (N) that adds 260 units of 1200A
9Primer.Before adding in preparation, primer should heat 7 minutes down at 65 ℃, and fast in cooled on ice.After adding enzyme and primer, final volume should use sterilized water to transfer to 200ml.The density of whole solution will be 1.14g/ml.
The final volume of the dosage that the per minute of reagent uniform solution is joined is usually little, 5-30 μ l for example, and preferred 10 μ l allow to produce the working volume of 10-100 μ l when being dissolved in the working solution when reagent formulation.
The uniform solution of the amount of pre-determining is dispensed into each hole of porous container, uses liquid to distribute automaton (robot) (96 holes/384 vent needles) usually.With 4 μ l to 20 μ l, but the volume of preferred 10 μ l distributes solution.
Drying means
Can be by the sample of lyophilization or lyophilization distribution.Suitable drying program produces has acceptable hardness, size, shape, T
g, porosity, dissolubility and stability reagent formulation.
The cryodesiccated overview of general success is shown in Table 1 below.
Table 1
Exsiccant method for optimizing is via lyophilizing.The reagent that distributes has carried out successful drying in 96 or 384 hole polystyrene plates.Surprisingly, when the polystyrene thin-walled part being placed the metallic plate support of outfit and being in direct contact with it, drying means carries out better and compares with exsiccant plate under the situation of no metal rack, does not observe the foam or the flocculus (Fig. 1 and 4) of exsiccant reagent.The direct contact area that has increased the metal shelf indirectly that contacts of the outer wall in polystyrene hole (pipe) and the grommet of metallic plate support, this has obtained better heat passage to sample conversely.On the other hand, for each hole, it is flat that the silicon dioxide mold has heavy wall pipe, and the lyophilization in the silicon dioxide mold is reasonably well carried out under the situation of not using metal rack.This may contact and the thermal transport property of silicon dioxide owing to mold and freeze dryer shelf better.Preferred freeze dried overview is shown in Table 2 below.Note, before carrying out follow procedure, sample under-46 ℃ in freeze dryer freezing 1 hour.
Table 2
Store
We have successfully prepared the stable biological reagent of tablet, cylinder and cubic form in based on the plate of polystyrene and silicon dioxide or mold.Our technology allowable temperature sensitive protein matter and nucleic acid molecules are stablized the stable at ambient temperature single agent form that changes into.Single agent form (pearl or cake) can comprise the required buffer agent of pre-assigned specific assay, salt, detergent and nucleotide etc., uses described molecule in described mensuration.These enzymes and combination can be used for many kinds of molecular biology uses, and includes but not limited to the synthetic application of PCR, RT-PCR, PCR in real time, whole genome amplification, in vitro transcription and cDNA.
Our method has been eliminated splashing in the liquid nitrogen or freezing needs to the cold surface reagent mixture is freezing, yet exsiccant reagent keeps good structural intergrity.Method has step still less in preparation process.When correct sealing, exsiccant reagent formulation can directly be stored in plate or the mold, and this has reduced preparation and packing cost significantly.Alternatively, can be used as tablet and take out exsiccant reagent from polyfiltronics 96 orifice plates, then it is stored in the bottle that the container of sealing is promptly added a cover from the silicon dioxide mold with as cubic block.
The sealing of plate or mold can be by following realization: medicated cap, band, hot activation band etc.In one embodiment of the invention, the sealing of plate seals by the hot activation of using AbGene ' s Thermo-Seal and Easy-Peel sheet and realizes.
Reagent formulation of the present invention is an ambient-temp-stable.About " ambient-temp-stable ", we are meant that preparation can be stored down at 22 ℃ and surpass 6 months, and it is compared with the activity of measuring behind the dried reagent in the first time, have to be less than 20% enzymatic activity and to lose.
Reagent formulation of the present invention has at least 10 ℃ glass transition temperature (T
g).The T that reagent formulation is general
gIt is 40 ℃.At least 40 ℃ T
gTo guarantee the stability under room temperature (22 ℃).Preferred T
gIt is 45 ℃.Glass transition temperature is such temperature, promptly is higher than this temperature, and the viscosity of glassy material reduces fast and glassy material is transformed into rubber, be transformed into then in addition higher temperature under be transformed into fluidic deformable plastics.
Glass transition temperature is as the important indicator of stability of formulation.Be lower than T
gTemperature or near at that time, sample remains stable glass.When rising, temperature is higher than T
gThe time, sample becomes rubber and unstable.T
gUse differential scanning calorimetry to measure.2-5mg (1-2 the ball of pulverizing) sample is placed aluminum pot.Make the sample experience determine the T of sample from 0 ℃ to 100 ℃ controlled temperature program by speed with 10 ℃/minute
gMeasurement flow to sample and is expressed as change (shift) the baseline from the effusive heat of sample and with it.T
gBe expressed as temperature at the mid point of this baseline change.
General porosity allows ball to be dissolved in the 20 μ l water in 1 minute or shorter time.Preferred porosity will allow to dissolve in 30 seconds or shorter time.
The method that we prepare with glass-faced (glassified) biological reagent preparation provides several advantages.Preparation method is simplified greatly.Reagent mixture directly is dispensed into the hole of porous plate, and described porous plate has the standard format identical with commercial microtitration plate.This has eliminated the needs that freezing reagent in liquid nitrogen or on cold surface is dripped.The mixture of freezing distribution then, lyophilizing in the hole, and also it can be stored in the hole.This has eliminated pearl or cake are transferred to the needs of hold-up vessel from dry pot.Because exsiccant compositions can be stored in the porous plate, so described compositions can be used in the subsequent working flow process of automatization.The robotics system can be used for disposable plates.
In addition, the compositions of preparation is stable at ambient temperature.This has saved cost of transportation (no dry ice transportation), has eliminated the needs that cold closet is stored and shortened the reagent preparation time (do not have and melt).It provides convenience for subsequent user.Great majority can be measured the dependency component allocates in advance and stablizes into single agent form.The plastics consumer goods cost that it has been saved user time and has been used to prepare the main mixture (master mix) of reagent.It also provides the repeatability and the reliability that increase, because it has reduced danger and the error polluted.Use pre-assigned reactant to prepare compositions.This make sample treatment and move the liquid step be reduced to minimum, thereby reduced the danger of the pollution that produces by the user and moved the liquid error.
Embodiment
Present embodiment only is provided for illustrational purpose, and should not be interpreted as limiting the scope of the present invention that is defined by the following claims.All lists of references that provide with other places below this description are all incorporated this paper into as a reference.
Embodiment 1: the preparation of the exsiccant PCR mixture that carries out in 384 hole polystyrene plates
Preparing the biological reagent preparation according to standard schedule, is the reagent mixture that is used for PCR in this situation.In brief, the preparation of 10 μ l balls comprises 25mM Tris-HCl (pH 9.0 is under room temperature), 125mM KCl, 3.75mM MgCl
2, 0.5mM dNTP, 0.6mg/ml BSA, the rTaq archaeal dna polymerase of 3.5 units and the packing material that comprises the formation glass of synthetic polymer Ficoll 400 (6.25%), Ficoll 70 (6.25%) and the second carbohydrate melezitose (10%).Before use usually with all reagent autoclaving or filtration sterilization.Preparation reagent and it is stored on ice until mixing and being dispensed into polystyrene board or silicon dioxide mold.Whole preparation is made up of the packing material that forms glass, BSA, dNTP and rTaq archaeal dna polymerase and above-mentioned salt.
Every dosage final volume of reagent uniform solution is 10 μ l.This allows the PCR working volume of 25 μ l.Use automatic pipettor that reagent is dispensed into 384 plate polystyrene board.
Place the top of cooling off the Vertis freeze dryer of (46 ℃) in advance to carry out coming in about 60 minutes freezing reagent 384 orifice plates.According to the method for describing in the aforementioned table 2, make refrigerated reagent experience first and second dry run subsequently then.
By with adhesive cover or medicated cap or apparatus for heat sealing (thermo seal) overlay the plate that comprises exsiccant reagent being stored in the resealable bag that comprises desiccant as integral packaging simply.Alternatively, exsiccant reagent is taken out as exsiccant reagent tablet or cubic block slave plate, then it is stored in the bottle, and bottle is stored in the reclosable bag that comprises desiccant.
By the PCR in real time amplification of λ DNA and will increase overview and commercial product (puRe TaqRTG pearl (beads); GE Healthcare) amplification overview is relatively tested the stability of exsiccant reagent composition.The primer that is used to test is SEQ ID NO:1 (5 '-GGT TAT CGA AATCAG CCA CAG CGC C-3 ') and SEQ ID NO:2 (5 '-GAT GAG TTC GTGTCC GTA CAA CTG G-3 ').PCR in real time amplification the results are shown in Fig. 2.
In Fig. 2,384 hole polystyrene plates are shown in the upper left side, and it is used to prepare exsiccant reagent composition.With exsiccant analoids developing agent storage at plastic bottle (lower-left side) with in the reclosable bag that comprises desiccant, storing about 1 week under the room temperature.384 orifice plates that comprise exsiccant reagent are shown in the upper left side, and it covers with the adhesive seal device.The standard curve of the different dilutions of λ dna profiling is shown in the upper right side.The real-time amplification curve of the different dilutions of λ DNA is shown in the lower right side.At last, the exsiccant reagent composition of successful proof of PCR in real time amplified reaction is stable.
Embodiment 2: the preparation of the exsiccant PCR mixture that carries out in 96 hole silicon dioxide molds
Mix according to the packing material of embodiment 1 preparation biological reagent preparation and formation glass and with them.Use automatic pipettor that about 20 μ l reagent mixtures are dispensed into 96 hole silicon dioxide molds (Fig. 3, upper left side).Place refrigerative in advance (46 ℃) Vertis freeze dryer to carry out coming in about 60 minutes freezing reagent the silicon dioxide mold.Make refrigerated reagent experience first and second dry run subsequently according to the method described in the aforementioned table 2 then.
Can be by covering with the adhesive seal device simply and by plate being placed the aluminum bag that comprises desiccant exsiccant reagent is stored in mold as integral packaging.Alternatively, take out exsiccant reagent from mold, then it is stored in the bottle that comprises desiccant as exsiccant reagent cake.
By according to the PCR in real time amplification of the λ DNA of embodiment 1 and will increase overview and commercial product (puRe Taq RTG pearl; GE Healthcare) amplification overview is relatively tested the stability of exsiccant reagent composition.The results are shown in Fig. 3.
In Fig. 3,96 hole silicon dioxide molds are shown in the upper left side, and it is used to prepare exsiccant reagent composition.Before carrying out λ DNA functional test, comprising in the reclosable bag of desiccant exsiccant reagent cubic block is stored in the plastic bottle (lower-left side) and carrying out for 1 week.The standard curve of the different dilutions of λ dna profiling is shown in the upper right side.The PCR in real time amplification of the different dilutions of λ is illustrated in the lower right side.In a word, the exsiccant reagent composition of successful proof of PCR in real time amplified reaction is stable.
Embodiment 3: the preparation of the exsiccant PCR mixture that carries out in 96 hole polyphenyl second plates
Mix according to the packing material of embodiment 1 preparation biological reagent preparation and formation glass and with them.Use liquid to distribute automaton to distribute 10 μ l reagent mixtures (Fig. 1, lower-left side).96 orifice plates are placed on the 96 mesoporous metal supports (Fig. 4).To place refrigerative in advance (46 ℃) Vertis freeze dryer to carry out coming in about 60 minutes freezing reagent with 96 orifice plates of metal rack.Make refrigerated reagent experience first and second dry run subsequently according to the method described in the aforementioned table 2 then.
Can be by with adhesive seal device or medicated cap or apparatus for heat sealing overlay under the help of heat sealing machine exsiccant reagent being stored in 96 orifice plates as integral packaging simply.Use freshly prepd " wetting " preparation and carry out the functional test of λ DNA PCR in real time with the exsiccant reagent of puRe Taq RTG pearl.The plate of sealing at room temperature or in 40 ℃ incubator was stored 8 days in the bag that comprises desiccant.PCR in real time amplification (according to embodiment 1) and will increase overview and commercial product (puRe Taq RTG pearl by λ DNA; GE Healthcare) amplification overview is relatively tested the stability of exsiccant reagent composition.The results are shown in Fig. 6,7 and 8.
Fig. 6 show λ qPCR that the PCR cake that uses 96 orifice dryings carries out and with the comparison of " wetting " preparation and pure Taq RTG pearl.This figure shows the stability according to the exsiccant PCR reagent of embodiment of the present invention preparation.Use starts from 1,000 ten thousand λ DNA that are copied to the variable concentrations of 10 copies and carries out the PCR in real time amplification as template together with the contrast of no template.For current form (upper right side), puRe Taq RTG pearl (lower left) and ' wetting ' preparation (upper left side), aspect Ct value and PCR efficient (bottom-right table), notice similar performance.
Fig. 7 shows the stability according to the exsiccant PCR reagent of embodiment of the present invention preparation.Exsiccant PCR mixture was stored 8 days down 40 ℃ or room temperature (RT), be used for the qPCR of λ DNA then.According to the reagent of the present invention's preparation, compare with commercial puRe Taq RTG pearl, obtain similar performance.The upper left corner: the exsiccant PCR reagent cake of under RT, storing.The upper right corner: in 40 ℃ of exsiccant PCR reagent cakes of storing down.The lower left corner: the puRe TaqRTG pearl that under RT, stores.The lower right corner: in 40 ℃ of puRe Taq RTG pearls that store down.Fig. 8 shows current form reagent and pure Taq RTG pearl Ct value and the PCR efficient under room temperature and 40 ℃.Embodiment 4: be used for the preparation of the exsiccant reagent of PCR in real time mensuration
PCR in real time becomes the more prevalent mensuration platform that is used for gene expression analysis.A method that is used for detecting the product that PCR in real time increases is used the homologous double labelling strand of specific part (ss) dna probe with template.Fluorescence on this probe is modified as reporter molecule (reporter) (FAM) and quencher (TAMRA).Under the situation that single-stranded template exists, probe and template annealing, but because reporter molecule dyestuff and quencher dyestuff tight adjacent and do not send fluorescence signal.When Taq archaeal dna polymerase during from template amplification DNA, the probe of the cutting of 5 ' of enzyme-3 ' exonuclease activity and the labelling of template annealing, thus discharge the quencher dyestuff, thus allow reporter molecule to fluoresce.Then by real-time instrument record fluorescence signal.
We prove that in this article PCR primer and TaqMan probe can carry out lyophilizing together under the situation that excipient exists, and do not carry out freeze dried reacting phase ratio with wherein primer and probe and can be used for PCR in real time and do not have loss function.The preparation that is used to prepare the 2.5x concentrate formulation that is used to measure beta-actin comprises: 25mM Tris pH 9,125mM KCl, 3.75mM MgCl
20.6mg/ml BSA, 0.5mM dNTPs, 0.25U/ μ l rTaq, 0.05%Tween 20,0.05%NP-40,1.5 μ M beta-actin forward primer (SEQ ID NO:3:5 '-TCA CCC ACACTG TGC CCA TCT ACG A-3 '), 1.5 μ M beta-actin reverse primer (SEQ IDNO:4:5 '-CAG CGG AAC CGC TCA TTG CCA ATG G-3 '), 1 μ M beta-actin probe (SEQ ID NO:5:5 '-FAM-ATG CCC-N (TAMRA) CCC CCATGC CAT C CTG CGT p-3 '), 6.25%Ficoll 70,6.25%Ficoll 400 and 10% melezitose.
The preparation of 10 mul aliquots samples is moved liquid go into 96 orifice plates, carry out lyophilizing then.Human gene group DNA's template of the amount of single exsiccant cake and variation is carried out rehydration in the final volume of 25 μ l.Use ABI 7900 Fast Real Time instrument to carry out real-time PCR reactions.Reaction is compared to the above-mentioned similar preparation that does not comprise primer and probe.For these after reaction, during PCR reaction is provided with, added primer and probe.In addition, the puReTaq RTG pearl of commercially available acquisition is as other contrast.
Fig. 9 shows the result of these reactions.The picture left above: use the freeze dried reagent PCR in real time of (comprising TaqMan primer and probe).Lower-left figure: freeze dried reagent contrast (primer and probe are not included in the freeze dried preparation, but add before real-time PCR reactions).Top right plot: commercial puReTaq RTG pearl contrast (every other reagent does not carry out lyophilizing).Generation amplification overview, the R of equal value of responding
2Value and slope.When puReTaq RTG reaction is used to produce standard curve and other reactions are considered as when unknown, the reaction with template DNA of equivalent drops on (bottom-right graph) on the standard curve.Our result proves that TaqMan primer and probe are easy to carry out RTG and integrate.
Embodiment 5: comprise the preparation of the exsiccant reagent of Phi29 archaeal dna polymerase
The Phi29 archaeal dna polymerase is widely used for whole genome amplification and rolling circle amplification.We carry out lyophilizing with this enzyme in making it possible to carry out the preparation of whole genome amplification.The freeze dried preparation of our analytical proof has in carrying out whole genome amplification and the identical activity of " wetting " preparation.
GenomiPhi HY (High Yield) DNA cloning test kit (GE Healthcare) comprises by isothermal strand displacement amplification and carries out the necessary all components of whole genome amplification.The raw material that is used for the GenomiPhi reaction can be the DNA of purification or the cell lysate of non-purification.The microgram amount of DNA can the raw material from the nanogram amount produces in a few hours only lacking.General dna output from GenomiPhi HY reaction is per 50 μ l reaction 40-50 μ g, and average product length is greater than 10kb.Because the check and correction 3 '-5 ' exonuclease activity of enzyme, dna replication dna is extremely accurate.
The GenomiPhi reactant mixture that will comprise Phi29 archaeal dna polymerase, random hexamer, dNTP and GenomiPhi HY reaction buffer and stabilizing agent Ficoll 70, Ficoll 400, melezitose and BSA prepares as the 2X mixture.The mixture of 10 μ l volume aliquots is dispensed into 12 hole PCR band pipes (strip tube).Use the VirTis freeze dryer that the product that distributes is lyophilized into cake.Exsiccant product was stored 35 days down room temperature or 40 ℃.Use is low to moderate the human gene group DNA of 10ng, successfully carries out whole genome amplification with these products.In the 0th time with after RT or 40 ℃ are stored 35 days down, this and the whole genome amplification that uses freshly prepd mixture to carry out are compared.
Figure 10 shows the result of the whole genome amplification mensuration that use freeze-dried reagent or " wetting " mixture carry out.The human gene group DNA of 10ng is used as mould material, under 30 ℃, carries out 90 minutes amplified reaction.Expection should produce the DNA greater than 4 μ g in 90 minutes.Use Pico Green to measure, promptly use and also detect intensive amplification at 40 ℃ of freeze dried reagent of storing 35 days down.The Phi29DNA polymerase with freeze dried form by stabilisation successfully.
Embodiment 6: be used for the preparation of the exsiccant reagent of in vitro transcription
Transcribing is the vital biological process of being undertaken by all types of cell routines, uses dna profiling in this process, and the RNA polymerase of utilization dependence DNA for example T7 RNA polymerase prepares RNA.This identical process of in vitro transcription (IVT) transcript that to be the generation carried out in the extracellular in test tube selected by the terminal use.The RNA molecule of gained can be used for proteinic external translation or for example RNA trace, southern blotting technique, microarray analysis and microinjection of hybridization then.
We have successfully produced freeze dried ambient temperature steady I VT reagent, and the RNA that wherein will remove template produces needed all components and carry out lyophilizing under the situation that excipient exists.This has greatly simplified the IVT reaction, thereby makes the terminal use only need add template DNA and water comes initial action.
The IVT preparation that is used to produce freeze dried reagent comprises 40mM Tris pH 8.0,10mMMgCl
2, 4mM spermidine, 10mM DTT, 50 μ g/ml BSA, 10mM NaCl, 0.5mMATP, 0.5mM CTP, 0.5mM GTP, 0.5mM UTP, 2U RNA Guard, 10UT7 RNA polymerase (GE Healthcare), 6.25%Ficoll 400,6.25%Ficoll 70,10% melezitose.The preparation of preparation is dispensed into 8 pore area pipes with the aliquot of 25 μ l, carries out lyophilizing according to embodiment 1 and table 2 then.Use is from the exsiccant reagent cake of contrast DNA tests of Roche SP6/T7 IVT test kit.Use the parallel IVT that carries out of Roche SP6/T7 IVT test kit to react.
As expected, use freeze dried reagent and Roche test kit to produce the transcript (Figure 11) of about 1000 base pairs.Therefore, transcribe necessary reagent under the situation that excipient exists by successfully lyophilizing, and it can carry out rehydration and produces the rna transcription thing in correct reaction volume under the situation that dna profiling exists.
Although shown and described the preferred embodiments of the invention, in this area, it should be apparent that, can be under situation without departing the teaching of the invention, change and modify.The mode that material shown in foregoing description and the accompanying drawing only illustrates by way of example provides rather than conduct limits.When considering in based on the correct viewpoint of prior art at them, actual range of the present invention is intended to limit in following claim.
Sequence table
<110>PONAKA，Reddy
FARCHAUS，Joseph?W.
PIERCE，Michael
＜120〉with the preparation of glass-faced biological reagent
<130>PB0689
<150>US?60/845,307
<151>2006-09-18
<150>US?60/887,364
<151>2007-01-31
<160>5
<170>PatentIn?version?3.3
<210>1
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>1
ggttatcgaa?atcagccaca?gcgcc 25
<210>2
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>2
gatgagttcg?tgtccgtaca?actgg 25
<210>3
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>3
tcacccacac?tgtgcccatc?tacga 25
<210>4
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<400>4
cagcggaacc?gctcattgcc?aatgg 25
<210>5
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic oligonucleotide
<220>
<221>misc_signal
<222>(7)..(7)
＜223〉be connected to the tetramethyl rhodamine
<220>
<221>misc_feature
<222>(7)..(7)
＜223〉n is a, c, g or t
<400>5
atgcccnccc?ccatgccatc?ctgcgt 26
Claims (18)
1. prepare the method for exsiccant reagent formulation, it comprises step:
(a) provide at least a aqueous solution through buffered biological reagent;
(b) will form the packing material of glass forms the concentration of packing material wherein and is enough to the mixture that promotes that glassy porous compositions forms with mixing through buffered reagent solution;
(c) mixture is dispensed in the hole of porous container with the form of dripping uniformly basically, wherein single is dispensed into each hole;
(d) dripping in the dry described container to form reagent formulation;
Wherein said reagent formulation is water miscible and has for room temperature stability is enough Tg.
2. the method for claim 1, it also comprises the reagent bottle that is collected into the storage of the prolongation that is used for exsiccant reagent with exsiccant.
3. the method for claim 1, it also comprises with the storage with the prolongation of carrying out exsiccant reagent of band or the sealing porous container of apparatus for heat sealing.
4. the method for claim 3, wherein said band is heat activated.
5. the process of claim 1 wherein that described porous container is the silicon dioxide mold.
6. the process of claim 1 wherein that described porous container is a polystyrene board.
7. the method for claim 6, wherein said polystyrene board is 96 orifice plates.
8. the method for claim 6, wherein said polystyrene board is 384 orifice plates.
9. the method for claim 6, it also comprises, before described drying steps, described polystyrene board is placed on the metal moulds, the outer wall in each hole of wherein said expanded polystyrene plate closely contacts with the hole of described metal moulds.
10. the process of claim 1 wherein that described drying steps realizes by lyophilizing.
11. the process of claim 1 wherein described at least a be to be used for the mensuration mixture measured biology through buffered biological reagent.
12. the method for claim 1, it also is included in the mixture of described drying steps freezing described distribution before.
13. comprise reagent composition according to the exsiccant reagent formulation of the method for claim 1 preparation.
14. the reagent composition of claim 13, wherein said exsiccant reagent formulation comprises enough biological reagents that is used for unitary determination.
15. comprising, the method for claim 11, wherein said mensuration mixture except amplification template and primer, be used for necessary all reagent of PCR.
16. comprising, the method for claim 11, wherein said mensuration mixture except template, be used for necessary all reagent of in vitro transcription.
17. comprising, the method for claim 11, wherein said mensuration mixture except template, be used for necessary all reagent of whole genome amplification.
18. the method for claim 11, wherein said mensuration mixture comprise that the PCR in real time that is used for except template measures necessary all reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510105699.2A CN104774924B (en) | 2006-09-18 | 2007-09-13 | Preparation of glass-coated biological reagents |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US84530706P | 2006-09-18 | 2006-09-18 | |
| US60/845,307 | 2006-09-18 | ||
| US60/887,364 | 2007-01-31 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510105699.2A Division CN104774924B (en) | 2006-09-18 | 2007-09-13 | Preparation of glass-coated biological reagents |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101516337A true CN101516337A (en) | 2009-08-26 |
Family
ID=41040407
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007800345077A Pending CN101516337A (en) | 2006-09-18 | 2007-09-13 | Preparation of glassified biological reagents |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101516337A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025892A (en) * | 2010-08-10 | 2013-04-03 | 凯杰有限公司 | Improved method for isothermal amplification of nucleic acids |
| CN104736726A (en) * | 2012-10-24 | 2015-06-24 | 通用电气医疗集团英国有限公司 | Direct nucleic acid amplification kit, reagent and method |
| CN105431527A (en) * | 2013-06-11 | 2016-03-23 | 比奥卡尔齐斯股份有限公司 | Biomolecule drying process for long-term storage |
| CN115176118A (en) * | 2019-12-16 | 2022-10-11 | 西北大学 | Lyophilized reagents |
-
2007
- 2007-09-13 CN CNA2007800345077A patent/CN101516337A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025892A (en) * | 2010-08-10 | 2013-04-03 | 凯杰有限公司 | Improved method for isothermal amplification of nucleic acids |
| CN104736726A (en) * | 2012-10-24 | 2015-06-24 | 通用电气医疗集团英国有限公司 | Direct nucleic acid amplification kit, reagent and method |
| US10907201B2 (en) | 2012-10-24 | 2021-02-02 | Global Life Sciences Solutions Operations UK Ltd | Direct nucleic acid amplification kit, reagent and method |
| CN112899354A (en) * | 2012-10-24 | 2021-06-04 | 通用电气医疗集团英国有限公司 | Direct nucleic acid amplification kit, reagent and method |
| CN105431527A (en) * | 2013-06-11 | 2016-03-23 | 比奥卡尔齐斯股份有限公司 | Biomolecule drying process for long-term storage |
| CN115176118A (en) * | 2019-12-16 | 2022-10-11 | 西北大学 | Lyophilized reagents |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104774924B (en) | Preparation of glass-coated biological reagents | |
| JP6097862B2 (en) | Compositions and methods for dehydrating and storing on-board reagents in microfluidic devices | |
| CN106661530B (en) | Microfluidic device | |
| US9790546B2 (en) | Microfluidic chip, device and system for the generation of aqueous droplets in emulsion oil for nucleic acid amplification | |
| US20080193946A1 (en) | Universal and Target Specific Reagent Beads for Nucleic Acid Amplification | |
| EP2703497B1 (en) | Microfluidic chip, device and system for the generation of aqueous droplets in emulsion oil for nucleic acid amplification | |
| CN108699593A (en) | Dry Amplification object | |
| US7217567B2 (en) | Method and formulation for lyophilizing cultured human cells to preserve RNA and DNA contained in cells for use in molecular biology experiments | |
| US11904315B2 (en) | System and self-metering cartridges for point of care bioassays | |
| CN101516337A (en) | Preparation of glassified biological reagents | |
| US20200318160A1 (en) | Sample to Sequence | |
| US20130280695A1 (en) | Method, device and test kit for molecular-biological reactions | |
| US20100015628A1 (en) | Ambient temperature stable chemical/biological reagents on membranes or filters | |
| US20220333178A1 (en) | Methods for improving nucleic acid cluster clonality | |
| US11898197B2 (en) | System and self-metering cartridges for point of care bioassays | |
| US20230193147A1 (en) | Wax-microsphere matrix compositions and methods of making and using the same | |
| CN105803534A (en) | Integrated sample preparation system and stable enzyme mixture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20090826 |