CN1918291A - Methods for nucleic acid isolation and kits using a microfluidic device and sedimenting reagent - Google Patents

Abstract

The present invention provides methods and kits for isolating nucleic acid from a sample, preferably from a biological sample, using a microfluidic device and sedimenting reagent.

Description

Use the method and the test kit of microfluidic device and sedimenting reagent isolating nucleic acid

Background of invention

Nucleic acid (for example, DNA and RNA) from composite interstitial substance such as blood, tissue sample, bacterial cell substratum and medical jurisprudence sample separate and purifying in genetic research, nucleic acid probe diagnostics, medical jurisprudence DNA check with to need other field of nucleic acid amplification be important method.The known multiple method for preparing amplification procedure with nucleic acid in this area, however each method all has each its limitation.

Relate to use density gradient separation peripheral blood lymphocytes (PBMC) from the most popular method of separation of whole blood DNA.Though this method is used effectively for research, it is not suitable for conventional integrated high-throughput microfluidic device usually.

The hypotonic buffer liquid that comprises the non-ionic type sanitising agent can be used for red corpuscle (RBC) and white corpuscle (WBC) cracking and keeps nucleus intact.In another approach, when the freeze thawing of whole blood process, has only the RBC cracking.Endorsing with by centrifugal recovery of undamaged WBC or they.For the RBC cracking is not destroyed WBC, can also make the use dilution as a kind of method.Other method that is used for the selective splitting of RBC comprises uses ammonium chloride or quaternary ammonium salt, and makes RBC through the hypo-osmoticity shock in the presence of hypotonic buffer liquid.Yet, in the ordinary method of one of these methods of use, the material of inhibition PCR (as, enzyme inhibitors) and nuclear and/or nucleic acid cosedimentation.Remove these inhibitor before must in the high-throughput microfluidic device of routine, analyzing.

Though for example boil, with the protease hydrolysis, be exposed to the extraction that processing under ultrasonic wave, sanitising agent or the highly basic has been used to DNA, alkaline extraction is one of the simplest method.For example, United States Patent (USP) 5,620,852 people such as () Lin described short reach at room temperature use in time of 1 minute alkali (as, NaOH) handle and effectively extract DNA from whole blood.Yet in order to obtain clean DNA, it is necessary removing dehemoglobinize and plasma proteins.This by using of short duration water-washing step to realize, for example by blood is suspended in the water, carries out centrifugal subsequently, abandoning supernatant, then with NaOH extract bead (pellet) (referring to for example, Biotechniques, Vol.25, No.4 (1998), the 588th page).Being used for a large amount of water of lysing cell makes this method not be suitable for the microfluidic device of standard.

United States Patent (USP) 5,010,183 people such as () Kellogg have been described the centrifugal microfluid based platform that uses alkaline lysis to extract DNA from blood.This method relate to primary sample (as, the whole blood or the E.Coli suspension of 5 microlitres (μ L)) mix with 10 milli (mM) NaOH that rub of 5 μ L, be heated to 95 ℃, keep 1-2 minute so that lysis, released dna and make protein denaturation to suppress PCR, by mixing with the 16mM TRIS-HCl (pH 7.5) of 5 μ L, the split product after the neutralization is mixed thermal cycling subsequently with liquid PCR reagent and the primer of 8-10 μ L with the split product neutralization.Regrettably, though amount of reagent is little and be suitable for microfluidic device, the downstream processing of DNA is still challenge in the microfluidic device.

Another ordinary method is used the phenol chloroform extraction.Yet it need use poisonous and have corrosive chemical, and is not easy to realize automatization.

Solid phase extractions also is used to separate nucleic acid.For example, from a method of nucleic acid source isolating nucleic acid relate to reaction vessel with the suspension of silicon dioxide granule with pass through buffered chaotropic agent such as guanidine thiocyanate and mix, add sample subsequently.In the presence of chaotropic agent, nucleic acid is adsorbed on the silicon-dioxide, makes it from liquid phase separation by centrifugal, washes with alcohol-water mixture, uses rare aqueous solution of buffer agent wash-out at last.The silicon-dioxide solid phase extractions need use pure washing step to remove residual chaotropic agent wash-out nucleic acid not; Yet must extreme care be used in the later step nucleic acid is increased or the susceptibility enzyme of modification to prevent to be suppressed at the alcohol of removing all traces (thermal evaporation or wash with another kind of very volatile and inflammable solvent).Water or elution buffer wash-out nucleic acid then.This combination, rinsing and elution process are the principles of many commercial reagent box, as Qiagen (Valencia, CA); Yet this method bothers and comprises a plurality of washing steps very much, makes it be difficult to be applicable to microfluidic device.

Ion exchange method produces high-quality nucleic acid.Yet ion exchange method causes the existence of high-caliber salt, must be removed before nucleic acid can be used further.

International open WO 01/37291A1 (MagNA Pure) described the application of magnetic glass particle and wherein by with the dedicated buffering liquid cultivation that comprises chaotropic salt and Proteinase K with the separation method of sample dissociation.Add the magnetic glass particle, be included in the surface that whole nucleic acid in the sample are incorporated into particle.Remove unconjugated material by the plurality of washing step.At last, at high temperature use the low salt buffer wash-out, obtain whole nucleic acid of purifying.

Also having another ordinary method to relate to is applied on the hydrophobic organic polymer solid phase biological sample optionally to hold back nucleic acid and to remove the nucleic acid that is trapped with nonionic surface active agent subsequently.Another method relates to nonionic surface active agent handles the hydrophobic organic polymer material, washing surface, and make treated SOLID ORGANIC polymer materials contact biological sample subsequently, be incorporated into the amount of the nucleic acid on the organic polymer solid phase with minimizing.Though these solid phase methods still need other method for being effective means from the biological sample isolating nucleic acid, particularly be fit to the method for microfluidic device.

Aforementioned discussion open and prior art do not constituted these data are admitted as the part of disclosed, known or common sense.

Summary of the invention

The invention provides the method that is used for isolating nucleic acid, preferred purifying and reclaims nucleic acid.Method of the present invention is used sedimenting reagent (that is sinking agent).Known sedimenting reagent is used for isolating the material that contains nucleic acid from inhibitor.Usually, inhibitor combines with sedimenting reagent and settles from sample, makes supernatant liquor comprise interested nucleic acid.Therefore, with after sedimenting reagent combines, sample comprises the enrichment region that contains most of interested nucleic acid and contains the inferior enrichment region of at least a portion sedimenting reagent (preferred most of sedimenting reagent) and at least a portion inhibitor (preferred most of inhibitor).

Isolating nucleic acid can be used in the test of the existence of concrete nucleic acid in the test sample for example according to the present invention.This test has importance in the prediction of disease and diagnosis, medical jurisprudence, epidemiology and public health.For example, separated DNA can be used for detecting the existence of individuality infective virus or mutator gene through hybridization and/or amplification, is used to measure the probability that this individuality suffers from the disease of communicable disease or genetics origin.Detect the infective virus in the sample in the screened hundreds of or thousands of samples or the ability of sudden change and significant importance is arranged for early diagnosis that is in the colony under the disease danger or epidemiology, for example, HIV infection, cancer or to the early detection of the susceptibility of cancer, or in neonatal general investigation of desease, wherein early detection has diagnosis of helping and treatment.In addition, method of the present invention can also be used for the fundamental research laboratory, is used for from cultured cells or biochemical reaction isolating nucleic acid.Nucleic acid can be used for enzymatically modifying such as digestion with restriction enzyme, order-checking and amplification.

The invention provides be used for from comprise nucleic acid (as, DNA, RNA, PNA) the method and the test kit of sample separation nucleic acid, described nucleic acid can be included in or be not included in and contain in the karyocyte (as, white corpuscle).These methods relate to from inhibitor such as protoheme (heme) and degraded product thereof (as, iron ion or its salt) final isolating nucleic acid, inhibitor is undesirable, because they can the suppression of amplification reaction (as, the situation in the PCR reaction).

Certain embodiments of the present invention relate to and remain on inhibitor in the solid phase material or remain on the nucleic acid that (that is, makes inhibitor be attached to this material) on the solid phase material and do not keep significant quantity.Suitable solid phase material generally include have connected catch the position (as, huge legendary turtle is closed functional group) any form (as, particle, protofibril, form membrane) solid substrate, be applied to coating reagent (preferred surfactant) on the solid phase material or both.

In one embodiment, the invention provides from the method for sample separation nucleic acid, this method comprises: the microfluidic device that comprises feed compartment, valve treatment chamber and mixing section are arranged is provided; Provide and comprise the material that contains nucleic acid and the sample of inhibitor; Sedimenting reagent is provided; Sample is placed feed compartment; With sample transfer to the valve treatment chamber is arranged; Use sedimenting reagent to form the sample concentration district in the valve treatment chamber is arranged, wherein the sample concentration district comprises that major part contains the material of nucleic acid, sample time enrichment region comprise at least a portion (with, be generally major part) sedimenting reagent and at least a portion inhibitor; Start the valve have in the valve treatment chamber at least a portion sample concentration district transferred to mixing section and to make at least a portion sample concentration district and the inferior enrichment region of sample separates; The material cracking (under optional heating) that will contain nucleic acid in mixing section is to discharge nucleic acid; With the optional pH that regulates the sample of the nucleic acid that comprises release.

In one embodiment, the invention provides from the method for sample separation nucleic acid, this method comprises: the microfluidic device that comprises feed compartment, valve treatment chamber and mixing section are arranged is provided; Provide and comprise material that contains nucleic acid and the sample of the cell that contains inhibitor (the described material that contains nucleic acid can be identical or different with the cell that contains inhibitor); Sedimenting reagent is provided; Sample is placed feed compartment, with sample transfer to the valve treatment chamber is arranged; Use sedimenting reagent to form the sample concentration district in the valve treatment chamber is arranged, wherein the sample concentration district comprises that major part contains the material of nucleic acid, and sample time enrichment region comprises at least a portion (with being generally most of) sedimenting reagent and at least a portion inhibitor; Start the valve have in the valve treatment chamber at least a portion sample concentration district transferred to mixing section and to make at least a portion sample concentration district and the inferior enrichment region of sample separates; The material cracking that will contain nucleic acid in mixing section is to discharge nucleic acid; With the optional pH that regulates the sample of the nucleic acid that comprises release.

If expectation, before the material cracking that will contain nucleic acid, this method can comprise that water (preferred not the sterilized water of qiagen rnase enzyme) or damping fluid dilute isolating sample concentration district, the zone that randomly further concentrates this process dilution with the concentration that increases nucleic acid substances, randomly separate and randomly repeat this dilution and concentrate subsequently to reach separating process and reduce to the concentration level that can not hinder amplification method with concentration inhibitor through further spissated zone.

Perhaps, if expectation, before the material cracking that will contain nucleic acid, simultaneously or afterwards, this method can comprise that isolating sample concentration district is transferred to the separate chamber is used to contact solid phase material, is attached to solid phase material preferentially to make at least a portion inhibitor; Wherein solid phase material comprise catch the position (as, huge legendary turtle is closed functional group), be applied to the coating reagent on the solid phase material or comprise both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.

The present invention also is provided for implementing the test kit of the whole bag of tricks of the present invention.

Definition

" nucleic acid " should have implication as known in the art, and be meant DNA (as, genomic dna, cDNA or plasmid DNA), RNA (as, mRNA, tRNA or rRNA) and PNA.It can be various ways, includes but not limited to bifilar or sub-thread structure, ring-type, plasmid, short relatively oligonucleotide, peptide nucleic acid(PNA) (being also referred to as PNA) (as people such as Nielsen, Chem.Soc.Rev. is 26, described in the 73-78 (1997)), or the like.Nucleic acid can be genomic dna, and it can comprise a complete karyomit(e) or a chromosomal part.DNA can comprise encoding sequence (as, mRNA, tRNA and/or rRNA are used to encode) and/or non-coding sequence (as, kinetochore, telomere, intergenic region, intron, transposon and/or microsatellite sequence).Nucleic acid can comprise any naturally occurring Nucleotide and carry out the Nucleotide of artificial or chemically modified, sudden change Nucleotide etc.Nucleic acid can comprise non-nucleic acid component, as peptide (as in PNA), marker (radio isotope or fluorescence labelling thing), or the like.

" material that contains nucleic acid " is meant the source of nucleic acid, as cell (as, the red corpuscle of white corpuscle, stoning), nucleus or virus or contain the structure that contains nucleic acid any other composition (as, plasmid, clay or viroid, archeobacteria).Cell can be protokaryon (as, gram-positive microorganism or Gram-negative bacteria) or eucaryon (as, hemocyte or histocyte).Be virus if contain the material of nucleic acid, it can comprise RNA or DNA genome; It can be fatal, attenuation or noninfectious; And it can infect protokaryon or eukaryotic cell.That the material that contains nucleic acid can be is naturally occurring, through manually modified or produce through artificial.

" isolating " is meant the isolated nucleic acid of at least a portion inhibitor from sample (that is at least a portion of at least a inhibitor) the material of nucleic acid (or contain).This comprises from other material such as cellular component such as protein, lipid, salt and other inhibitor isolates required nucleic acid.More preferably, isolating nucleic acid is purifying basically." purifying basically " is meant separation at least 3 piks/microlitre (pg/ μ L), preferred at least 2 nanograms/microlitre (ng/ μ L), more preferably the nucleic acid of 15ng/ μ L at least makes the amount of inhibitor in the primary sample reduce at least 20% simultaneously, preferred reduction at least 80% more preferably reduces at least 99%.Impurity is cellular component and the nuclear components the solvent in sample normally, as protoheme and associated products (teichmann's crystals, protoheme) and metal ion, protein, lipid, salt etc.Therefore, term " purifying basically " generally be meant from sample separation go out most of inhibitor (as, protoheme and degraded product thereof), make and remove the compound of using subsequently that can hinder isolating nucleic acid at least in part.

" be attached to " or " adhering to " or " combination " is meant that inhibitor is detained the reversibility of optional solid phase material, it can pass through number of mechanisms, comprises more weak power, holds back as Van der Waals interaction, electrostatic interaction, affinity combination or physics.The use of this term does not hint mechanism of action, and comprises absorption mechanism and mechanism of absorption.

" solid phase material " is meant inorganic or organic materials, the preferred polymkeric substance of making by repeating unit, and repeating unit can be identical or different, is the compound in organic and/or inorganic, natural or synthetic source.This comprise homopolymer and heteropolymer (as, copolymer, terpolymer, tetrapolymer etc., it can be for example random or block).This term comprises the polymkeric substance of fibrous or particle form, and it can easily prepare by means commonly known in the art.This material forms porous matrix usually, though for some embodiment, solid phase also refers to solid surface, as the atresia sheet material of polymer materials.

Optional solid phase material can comprise catches the position." catch the position " and be meant the position that is used for attachment material on the solid phase material.Usually, catch the position comprise and be covalently attached to or otherwise be connected in (as, hydrophobicity is connected in) functional group or molecule on the solid phase material.

Phrase " is applied to the coating reagent on the solid phase material " and is meant the material at least a portion that is coated on solid phase material, as, be coated on the material at least a portion of fibril-matrix and/or adsorptivity particle.

" tensio-active agent " is meant that reducing it is dissolved in the material of the surface tension or the interfacial tension of medium wherein.

" highly basic " is meant complete dissociated alkali in water, as, NaOH.

" polyelectrolyte " is meant the ionogen as electropolymer, and it has usually than higher molecular weight, as polystyrolsulfon acid.

" the polymkeric substance barrier of alternative infiltration " is meant the polymkeric substance barrier according to size and the alternative transhipment of electric charge liquid.

" enrichment region " is meant the bead form that can be, and has the sample area of the material that contains nucleic acid, nucleus and/or the nucleic acid of higher concentration with respect to inferior enrichment region.

As used in this article, " separate basically ", particularly in sample concentration district and the sample time isolating situation of enrichment region, be meant remove the sample total amount be lower than at least 40% of 25% nucleic acid (no matter it is for free or in nucleus or in other contains the material of nucleic acid) total amount.Preferably, be lower than at least 75% of 10% nucleic acid total amount from what the rest part of sample was isolated the sample total amount.More preferably, be lower than at least 95% of 5% nucleic acid total amount from what the rest part of sample was isolated the sample total amount.

" inhibitor " is meant and is used for for example enzyme inhibitors of amplified reaction.The example of this inhibitor generally includes iron ion or its salt (as, Fe ²⁺Or its salt) and other metal-salt (as, alkalimetal ion, transition metal ion).Other inhibitor can comprise protein, peptide, lipid, carbohydrate, protoheme and degraded product thereof, urea, bile acide, humic acid, polysaccharide, cytolemma and kytoplasm component.The main inhibitor of PCR in human blood is oxyphorase, lactoferrin and IgG, and it is present in respectively in red corpuscle, white corpuscle and the blood plasma.Method of the present invention makes at least a portion inhibitor (that is at least a portion of at least a inhibitor) and the separating substances that contains nucleic acid.As discussing herein, contain inhibitor cell can with contain nuclear cell or other material that contains nucleic acid is identical.Inhibitor can be included in the cell or can be positioned at the extracellular.The extracellular inhibitor comprises that all are not included in intracellular inhibitor, and it comprises those inhibitor that for example are present in serum or the virus.

The inhibitor that " preferentially makes at least a portion inhibitor be attached to solid phase material " to be meant one or more types than the material that contains nucleic acid (as, nucleus) and/or nucleic acid be attached on the optional solid phase material with bigger degree, and usually the material that contains nucleic acid and/or the nucleus of signal portion are attached on the solid phase material.

" microfluid " is meant the device with one or more fluid channels, chamber or conduit, its at least one inner cross-sectional dimension that has (as, the degree of depth, width, length, diameter etc.) less than 500 μ m, be generally 0.1 μ m to 500 μ m.In the device that the present invention uses, at least one cross-sectional dimension of preferred microscale channel or chamber be 0.1 μ m to 200 μ m, more preferably 0.1 μ m to 100 μ m, be generally 1 μ m to 20 μ m.Usually, microfluidic device comprises a plurality of chambers (treatment chamber, separate chamber, mixing section, waste chamber, thinner chamber, amplified reaction chamber, feed compartment etc.), and each chamber is defined for the capacity that comprises sample; And there is at least one to distribute passage to connect a plurality of chambers of array; For example, wherein at least one chamber in the array can comprise that at least one chamber in solid phase material (thereby it often is known as the separate chamber) and/or the array can comprise cracking agent (thereby it often is known as mixing section).

Term " comprises " and variant is not to have restrictive sense when these terms appear in specification sheets and the claim.

As used in this article, " one ", " one ", " being somebody's turn to do ", " at least one " and " one or more " are used interchangeably and are meant one or more.

Also in this article, the narration of the numerical range of representing by end points comprise all numbers of being comprised in this scope (as, 1 to 5 comprises 1,1.5,2,2.75,3,3.80,4,5 etc.).

Foregoing invention content of the present invention is not used in describes each disclosed embodiment or all execution of the present invention.Following description more specifically illustrates exemplary embodiment.Several places in the application provide guidance by enumerating of embodiment, and described embodiment can use by various combination.In each case, it is just representational to enumerate example, it should be interpreted as the exclusive example of enumerating.In addition, described different embodiments, wherein a plurality of elements of each embodiment can be used to other embodiment, even without describing particularly.

Description of drawings

Fig. 1 represents to be used for the microfluidic device of some method of the present invention.

The detailed description of exemplary

The invention provides and be used for, biological sample normally, isolating nucleic acid, the preferred several different methods and the test kit of the nucleic acid of pure form basically from sample.The invention provides be used for from comprise nucleic acid (as, DNA, RNA, PNA) the method and the test kit of sample separation nucleic acid, described nucleic acid can be included in or be not included in and contain in the karyocyte (as, white corpuscle).

Should be appreciated that though this method relates to from sample separation nucleic acid, this method may not be to remove nucleic acid from the material that contains nucleic acid (as, nucleus).That is to say, may need other step, with further from for example nucleus isolating nucleic acid.

Method of the present invention relates to finally makes nucleic acid and inhibitor such as protoheme and degraded product thereof (as, molysite) separate, and inhibitor is undesirable, but because the reaction of their suppression of amplification (as, the situation in the PCR reaction).More specifically, method of the present invention relates at least a portion nucleic acid that makes in the sample and separates with at least a portion of at least a inhibitor.Preferable methods relates to all basically inhibitor of removing in the sample that comprises nucleic acid, makes that nucleic acid is pure basically.For example, the ultimate density of ferruginous inhibitor is not more than about 0.8 micromole (μ M), and it is the permissible level that exists in the conventional PCR system.

In order to obtain clean DNA from whole blood, expectation removes dehemoglobinize and plasma proteins usually.When erythrocyte splitting, discharge protoheme and related compound, they suppress the Taq polysaccharase.Normal hemoglobin concentration is per 100 milliliters (mL) 15 grams (g) in the whole blood, based on this, is about 10 millis rub (mM) through the concentration of the protoheme in the hemolytic whole blood.For PCR is carried out satisfactorily, the concentration of protoheme should be reduced to micromole (μ M) level.This can realize by dilution, or for example remove inhibitor with inhibitor bonded material by use and realize.

Usually, the sample that will comprise nucleic acid is handled in circulation (flow-through) container, though this container is not essential requirement of the present invention.Preferably, for some method of the present invention, treatment facility is a microfluidic structures.

Method of the present invention is used sedimenting reagent (that is sinking agent).Known sedimenting reagent is used to make the material that contains nucleic acid to separate with inhibitor.Usually, inhibitor combines with sedimenting reagent and settles from sample, makes supernatant liquor comprise interested nucleic acid.Therefore, with after sedimenting reagent combines, sample comprises the enrichment region that contains most of interested nucleic acid and contains the inferior enrichment region of at least a portion sedimenting reagent (preferred most of sedimenting reagent) and at least a portion inhibitor (preferred most of inhibitor).

Sedimenting reagent can be dextran or load the gelatin of ZeptoGel salt (ZeptoMetrixCorporation, Buffalo, NY).Sinking agent can be added in the dry structure and be stored in the microfluidic device and add entry up to the user, is used for for example producing 6% solution, adds sample (as, blood) subsequently.In other situation, can sedimenting reagent and sample be joined in the microfluidic device together by the user.Make then the mixture sedimentation a little while (as, continue to many 45 minutes, though can use the longer time in some cases).If sample is a blood, the supernatant liquor that will be rich in lymphocyte (white corpuscle) then is separated in another chamber, makes to separate from the sediment that is rich in red corpuscle (red blood corpuscle).Usually will be rich in lymphocytic layer then and carry out cracking, remove the inhibitor of these releases subsequently to destroy any residual red blood cell contamination thing.

In some cases, the supernatant liquor that is rich in lymphocyte (white corpuscle) can comprise inhibitor (as because part haemolysis).These inhibitor can be removed or remove by a series of concentrating/separation/optional dilution step by using solid phase material.

Sample

Method of the present invention can be used for from various samples, biological sample particularly, isolating nucleic acid, described sample such as body fluid (as, whole blood, serum, urine, saliva, cerebrospinal fluid, seminal fluid or synovia lymph liquid), different tissues (as, skin, hair, tongue fur, ight soil, knurl or organ such as liver or spleen), cell culture or cell culture supernatant liquid etc.Sample can be foodstuff samples, drink sample, fermenting broth, the diagnosis that is used for disease or illness or disposal or monitoring or treatment clinical sample, medical jurisprudence sample, agriculture sample (as, derive from plant or animal) or environmental sample (as, soil, dirt or rubbish).

Biological sample is to have biogenetic derivation or biochemical those of originating.Those biological samples that are suitable in the inventive method can be from Mammals, plant, bacterium or yeast source.Biological sample can be independent cells form or is organizational form.Cell or tissue can be from isolated culture.Importantly be, certain embodiments of the present invention use without any pre-treatment (as, cracking, filtration etc.) whole blood as interested sample.

For some embodiment, sample such as whole blood can carry out pre-treatment by centrifugal, and from blood separation white corpuscle (that is buffy coat) and as the sample in the inventive method.

For some embodiment, sample can experience ultracentrifugation with concentrating sample before it experiences processing of the present invention.

Sample can be dissolving or is dispersed in solid sample in water or the organic medium (as, solid tissue), or therefrom with the solid sample of nucleic acid extraction in water or the organic medium.For example, sample can be organ homogenate (as, liver, spleen).Therefore, sample can comprise the nucleic acid (if particularly it is solid sample) through extracting in advance.

The type of sample is not restriction of the present invention.Yet, usually, sample comprise the material that contains nucleic acid and need with the inhibitor of separate nucleic acid.In this context, the material that contains nucleic acid be meant various cells (as, white corpuscle, bacterial cell), nucleus, virus or contain the structure that contains nucleic acid any other composition (as, plasmid, clay or viroid, archeobacteria).In some preferred embodiment of this method, the material that contains nucleic acid comprises nucleus.

In certain embodiments, sample can be the part cracked (as, pre-cracking is with release inhibitor, for example water carries out erythrocytic cracking), in this case, may need cracking in the method for the invention; Yet, usually, the fully not pre-cracking of the sample that contacts with sedimenting reagent (or preferably, or even partly pre-cracking).For example, when contacting with sedimenting reagent when strengthening red corpuscle and inhibitor wherein and settle, red corpuscle should be preferably undamaged (that is, not destroyed).Yet, can there be some to mix with white corpuscle in the supernatant liquor sometimes from the inhibitor in the ruined red corpuscle, it can use other technology to remove then.

Isolating (promptly, isolating with inhibitor) nucleic acid can be used for (preferably without being further purified or washing) multiple application (as, amplification, order-checking, mark, cancellation, digestion with restriction enzyme, connection, reversed transcriptive enzyme, hybridization, southern blotting technique, RNA trace etc.).Particularly, it can be used for measuring the genome of main body.It can be used for diagnosing microorganism in the sample (as, bacterium, virus) existence, and can be used for monitoring and/or treating the damage that causes by microorganism subsequently to sample source.Method of the present invention, material, system and test kit be particularly suitable for preparation high-throughput or automatization handle use in (particularly microfluid system) amplification technique (as, PCR, LCR, MASBA, SDA and bDNA) use nucleic acid extractive.Therefore, for certain embodiments of the present invention, isolating nucleic acid is transferred in the amplified reaction chamber (as the PCR sample chamber in the microfluidic device).

Can be according to the present invention from impure, partial-purified or pure sample separation nucleic acid (that is, separating) with inhibitor.The purity of primary sample is not vital because can from addition very impure sample separation go out nucleic acid.For example, can obtain nucleic acid from impure biologicfluid sample such as blood, saliva or tissue.If the primary sample of expectation higher degree, can make sample before experience method of the present invention according to well known to a person skilled in the art that any ordinary method handles.For example, can handle sample, make and before sample experiences method of the present invention, remove some impurity, as insoluble substance.

Isolating nucleic acid as described herein can have any molecular weight and be sub-thread form, bifilar form, annular, plasmid etc.Can multiple nucleic acid is separated from one another (as, from the DNA isolation of RNA, or separate double-stranded DNA from single stranded DNA).For example, can use method of the present invention to separate to have about 10 little oligonucleotide or nucleic acid molecule, have about 1000 bases to the longer molecule of about 10,000 base length even have the macromolecule nucleic acid that about 50kb arrives about 500kb to about 50 base length.In certain aspects, isolating nucleic acid can preferably have about 10 bases to about 100,000 bases according to the present invention.

The sample that contains nucleic acid can have different capacity.For example, for microfluidic structures, usually preferred very little capacity, as, 10 μ L (preferably being not more than 100 μ L).Should be appreciated that,, can use more jumbo sample if concentrate as passing through through pre-treatment.

For the low copy number gene, need bigger sample capacity to be present in the sample usually to guarantee interested sequence.Yet bigger sample size has more substantial inhibitor, and makes usually and himself be unsuitable for microfluidic structures.Therefore, for the situation of low copy number, may need to use 100 μ L or bigger capacity, to obtain reproducible result; Yet because higher sample capacity, the handled sample number of each microfluidic device may reduce.

In some method of the present invention, separating enrichment region (as, the supernatant liquor of lymphocyte-rich) afterwards, the centrifugation step that is used for concentrating the material that contains nucleic acid is useful for the low copy number sample.Yet though nucleic acid concentration significantly increases in for example bottom of treatment chamber, inhibitor concentration is still higher.Though in inferior enrichment region, remove most of inhibitor, be among serum and the ruined RBC protein (as, protoheme and protoheme associated products), but the enrichment region that contains nucleic acid of sample still has the inhibitor of significant quantity to exist, yet, nucleic acid is very high with the ratio of inhibitor, produces the spissated relatively sample of nucleic acid.Then, if desired, can make the optional solid phase material of this sample concentration district contact or through a series of concentrating/separate/optional dilution step, as described herein, to remove residual inhibitor (usually before cracking).

For the high copy number gene, can use the sample capacity that reaches 2 μ L for a short time, but be to use more large vol when (as, 20 μ L) circulation ratio better.In situation, can obtain higher throughput (that is the sample number of each microfluidic device processing) than low capacity.In the comparatively large capacity situation of (as, 20 μ L), may need not experience the pre-rotation step that is used for concentrating the cell that contains nucleic acid.

For those embodiments of wherein also using solid phase material except sedimenting reagent, the sample that contains nucleic acid that is applied on the solid phase material can be any amount, and this amount is by the amount decision of solid phase material.Preferably, the amount that is applied to the sample amplifying nucleic acid on the solid phase material is lower than the dry weight of solid phase material, is generally about 1/10,000 to about 1/100 (weight, nucleic acid/solid phase).The amount that is applied to the sample amplifying nucleic acid on the solid phase material can for example reach 100 grams or low 1 pik that reaches.

Preferably from the isolating required nucleic acid of the inventive method for the sample that applies at first all at least 20% of the amount of nucleic acid, more preferably at least 30%, more preferably at least 70%, most preferably at least 90%.Therefore, some preferred method of the present invention provides with high-recovery and reclaims required nucleic acid from sample.In addition, can reclaim the nucleic acid molecule of minute quantity quantitatively according to the present invention.The rate of recovery or yield depend primarily on the quality rather than the method itself of sample.Do not need from the spissated nucleic acids for preparation method of large vol because certain embodiments of the present invention provide, so the present invention has avoided the danger of nucleic acid loss.

Having too many DNA in the PCR sample may be harmful to the amplification of DNA, because there is too many mistake to draw position (misprimed site).The non-target sequence that this produces many linearities or increases by exponential manner.Because the specificity of amplification disappears along with the increase of non-target dna amount, the index with any significance degree generation target sequence does not gather.Therefore, wish the amount that control enters the DNA in each PCR sample.The amount of DNA is no more than 1 microgram/reaction usually, is generally at least 1 pik/reaction.Typical final DNA concentration is that 0.15 nanogram/microlitre is to 1.5 nanograms/microlitre in the PCR mixture.In the situation of microfluidic device, can make each sample have an amount of DNA with the sample shunting after purifying, before the PCR.Perhaps, sample sufficiently can be diluted in the sample processing device that comprises the treatment chamber that variable valve is housed as more specifically described as the following (being specially microfluidic device), make to have an amount of DNA in each PCR mixture.In diagnostic device, because leukocytic amount can be significantly different, the amount of the DNA that very difficult ex ante forecasting will be separated.Yet useful range is that per 200 μ L blood contain the DNA of 3 micrograms (μ g) to 12 μ g.For buffy coat, useful range is that per 200 μ L buffy coats contain the DNA of 25 μ g to 50 μ g.

Lytic reagent and cracking condition

For certain embodiments of the present invention, some time points in treating processes are with the lysis in the sample, particularly contain nucleic acid cell (as, white corpuscle, bacterial cell, virocyte), with the content that discharges cell and form sample (that is split product).The physics of the film that is cracked into each cell of this paper breaks, be meant the okioplast film and when having nuclear membrane the physics of nuclear membrane break.This can use standard technique to carry out, as make the hot deactivation of proteolytic enzyme subsequently by protease hydrolysis; With tensio-active agent (as, nonionic surface active agent or sodium lauryl sulphate), guanidinesalt or highly basic (as, NaOH) handle; Physical damage (as, use ultrasonic wave); Boil; Or heating/cooling (as, be heated to minimum 55 ℃ (usually to 95 ℃) and cool to room temperature or following (to 8 ℃)), it can comprise the freeze/thaw processing.Usually, if use cracking agent, it is in the water medium, though if can be with an organic solvent during expectation.

Usually, after sample being contacted sedimenting reagent and separating more spissated zone, make the agent of sample contact cracking.Cracking agent can be nonionogenic tenside, for example is used to discharge nucleus.

White corpuscle can use the tensio-active agent cracking to produce no undamaged nucleus.Nonionic surface active agent such as TRITON X-100 can be joined in the TRIS damping fluid that contains sucrose and magnesium salts, be used for nuclear separation.

The amount that is used for the cracked tensio-active agent is enough high, with effective lysate sample; Also enough low, for example precipitate avoiding.The surfactant concentrations that is used for cracking process is generally at least 0.1 weight % based on the gross weight of sample.The surfactant concentrations that is used for cracking process is not more than 4.0 weight % usually based on the gross weight of sample, preferably is not more than 1.0 weight %.Usually optimize this concentration to reach complete cracking in the shortest as far as possible time, the mixture that obtains is that PCR is compatible.In fact, the nucleic acid that joins in the preparation in the PCR cocktail should seldom suppress or suppress hardly PCR in real time.

If expectation can mix use with damping fluid with tensio-active agent.Usually, sort buffer liquid provides pH to be at least 7, is up to 9 sample usually.

Usually, can use even stronger cracking agent, as highly basic, with any white corpuscle of cracking to discharge nucleus.For example, United States Patent (USP) 5,620, the method that 852 people such as () Lin describe can be suitable for some method of the present invention, its relate at room temperature use alkaline purification (as, NaOH) extract DNA from whole blood short reaching in time of 1 minute.Usually, have multiple highly basic to can be used in the alkaline lysis to produce effective pH (as, 8-13, preferred 13).Highly basic is generally oxyhydroxide, as NaOH, LiOH, KOH; Has the quaternary nitrogen containing positively charged ion oxyhydroxide of (as, quaternary ammonium); And alkali such as tertiary amine, secondary amine or primary amine.Usually, alkaline concentration is at least 0.01 equivalent concentration (N), and is up to 1N usually.Usually, then with mixture neutralization, if nucleic acid experience amplification procedure (as PCR) subsequently particularly.Therefore, certain embodiments of the present invention comprise regulate sample pH usually to minimum be 7.5, and be not more than 9 usually.In another approach, can after with alkaline lysis, heat, subsequently sample be neutralized further to make protein denaturation.

Can also under heating, use Proteinase K, under higher temperature, make the hot deactivation of Proteinase K subsequently, be used for from nucleus or WBC isolating nucleic acid.

Can also use commercially available cracking agent and neutralizing agent, as Extract-N-Amp Blood PCR test kit according to the Sigma of microfluid dimensions scale downward.(Oslo, POWERLYSE Norway) are used to make be difficult to cracked bacterium such as cracking such as staphylococcus, suis, to help some method of the present invention as deriving from GenPoint can to use stronger cracked solution.

In another approach, can use boiling method lysing cell and nucleus, released dna, and precipitate oxyphorase simultaneously.DNA in the supernatant liquor need not enrichment step can be directly used in PCR, makes this method can be used for the low copy number sample.

Optional solid phase material

For certain embodiments of the present invention, can use solid phase material (rather than sedimenting reagent).For example, sedimenting reagent can be joined in the blood, allow RBC to settle.Supernatant liquor (separated portions) comprises nucleic acid substances (in WBC), hemolytic inhibitor takes place (from a part by the RBC of water-splitting) and serum protein.Can make this separated portions contact solid phase material then, hemolytic RBC (as, ferruginous inhibitor) take place to remove.Subsequently with the WBC cracking to discharge nucleic acid.

Have been found that inhibitor is attached to solid phase (preferred polymers) material, this material comprise any form (as, particle, protofibril, form membrane) solid substrate, preferably it has the connected position (as, chelating functional group) of catching; Be applied to the coating reagent (preferred surfactant) on the solid phase material; Or have both.Coating reagent can be positively charged ion, negatively charged ion, nonionic or amphoteric ionic surfactant.Perhaps, coating reagent can be polyelectrolyte or highly basic.If expect, can use the various combination of coating reagent.

The solid phase material that can be used for method of the present invention can comprise the multiple organic and/or inorganic materials that for example keeps inhibitor such as protoheme and hemachrome degradation product (particularly iron ion).This material with catch position (preferred chelation group) functionalized, scribble one or more coating reagents (as, tensio-active agent, polyelectrolyte or highly basic) or both.Usually, solid phase material comprises organic polymer matrix.

Usually suitable material is chemically inert, physics and chemically stable and compatible with multiple biological sample.The example of solid phase material comprise silicon-dioxide, zirconium white, alumina bead, metallic colloid as gold, for example by the sulfydryl chemistry carry out functionalized processing be used to produce catch the position cover the gold plaque material.The example of suitable polymkeric substance comprises for example polyolefine and fluorinated polymer.Usually before using, the solid phase material washing is desalted and other impurity to remove.It can stored dry or is stored in the aqeous suspension standby.Preferred solid phase material is used for the container that circulates, such as in for example transfer pipet, syringe or stake, microtiter plate or the microfluidic device, though can also use the suspension method that does not relate to these containers.

The solid phase material that can be used for method of the present invention can comprise the multiple material of various ways.For example, it can be particle loose or that be fixed or globule form, fiber, foam, frit, microporous membrane, film or has the substrate form on little duplicating (microreplicated) surface.If solid phase material comprises particle, preferably they be uniformly, globular and inflexible, to guarantee good fluid flow characteristics.

Use for circulation of the present invention, this material is generally loose porous network form, is used to allow macromole to pass in and out equably and with being without prejudice, and is used to the surface-area that provides bigger.Preferably, for this application, solid phase material has than higher surface area, such as for example, surpasses 1 meters squared per gram (m ²/ g).For not relating to the application of using fluid means, solid phase material can the yes or no porous matrix.Therefore, film also can be used in some method of the present invention.

For the application of using particle or globule, they can be incorporated in the sample or sample be incorporated in the bed of particle/globule and and therefrom remove by for example centrifugal.Perhaps, can pass through several different methods (as, spraying drying) with particle/globule coating (as, figure coating) to the inert substrate that randomly scribbles tackiness agent (as, polycarbonate or polyethylene) on.If expectation can be duplicated substrate is little, be used to increase surface-area and strengthen and purify.Can also carry out pre-treatment with oxygen plasma, electron beam or uv-radiation, heating or corona treatment method.Substrate can be used as for example mulch film on the bank of microfluidic device, or is laminated on the mulch film.

In one embodiment, solid phase material comprises fibril-matrix, and it can have or not be absorbed in wherein particle.Fibril-matrix can comprise any in the multiple fiber.Usually, fiber is insoluble at aqueous environment.Its example comprises glass fibre, polyolein fiber (particularly polypropylene and polyethylene primitive fiber, Kevlar, fluorinated polymers fibres (particularly polytetrafluoroethylene fiber) and natural cellulosic fibre.Can use the mixture of fiber, its combination for nucleic acid can be active or inactive.Preferably, fibril-matrix formation has at least about 15 microns and is about 1 millimeter to the maximum, more preferably is the tablet of about 500 micron thickness to the maximum.

If the use particle, then particle is normally insoluble in aqueous environments.They can be made by the combination of a kind of material or multiple material, as in the coating particle.They can be expandable or nondistensible, though preferably they are nondistensible in water and organic liquid.Preferably, if particle is used to adhere to, it is made by non-bloating hydrophobic material.They can be selected according to the affinity to nucleic acid.The example of the expandable particle of some water is at United States Patent (USP) 4,565, describes among 663 people such as () Errede, 4,460,642 people such as () Errede and 4,373,519 people such as () Errede.Nondistensible particle is at United States Patent (USP) 4,810 in water, describes among 381 people such as () Hagen, 4,906,378 people such as () Hagen, 4,971,736 people such as () Hagen and 5,279,742 people such as () Markell.Preferred particle is a polyolefin particles, as polypropylene particles (as, powder).Can use the mixture of particle, its combination for nucleic acid can be active or inactive.

If use the coating particle, coating is preferably water insoluble and be insoluble to the nondistensible material of organic solvent.Coating can be adhered to or non-cohesive nucleic acid.Therefore, the fundamental particle of coating can be inorganic or organic.Fundamental particle can comprise the inorganic oxide that its covalent attachment is had organic group, as silicon-dioxide, aluminum oxide, titanium dioxide, zirconium white etc.For example, can use covalently bound organic group, as have the aliphatic group of different chain length (C2, C4, C8 or C18 group).

The example of suitable solid phase material that comprises fibril-matrix is at United States Patent (USP) 5,279,742 (people such as Markell), 4,906,378 (people such as Hagen), 4,153,661 (people such as Ree), 5,071,610 (people such as Hagen), 5,147,539 (people such as Hagen), 5,207,915 (people such as Hagen) and 5, describe among 238,621 people such as () Hagen.This material can be available from 3M Company (St.Paul, MN), trade name is SDB-RPS (Styrene-Divinyl Benzene Reverse PhaseSulfonate, 3M Part No.2241), positively charged ion-SR film (3M Part No.2251), C-8 film (3M Part No.2214) and negatively charged ion-SR film (3M Part No.2252).

Preferably include those of tetrafluoroethylene matrix (PTFE) especially.For example, United States Patent (USP) 4,810,381 (people such as Hagen) disclose solid phase material, it comprises: the tetrafluoroethylene fibril-matrix, with the nondistensible adsorptivity particle that is trapped in the matrix, the weight ratio of wherein nondistensible particle and tetrafluoroethylene is about 19: 1 to 4: 1, and other wherein compound solid phase material has the clean surface energy of 20 to 300 milli Newton/meter.United States Patent (USP) RE 36,811 (people such as Markell) disclose the solid phase extractions medium, it comprises: fibril-matrix, with the adsorptivity particle that is trapped in the matrix, wherein particle comprises the porousness organic filler above 30 to maximum 100 weight %, with the porousness that is less than 70 to 0 weight % (applying organic layer or uncoated) inorganic particulate, the weight ratio of adsorptivity particle and PTFE is 40: 1 to 1: 4.

Particularly preferred solid phase material derives from 3M Company, and St.Paul is under the trade name EMPORE of MN.The ultimate principle of EMPORE technology is to use any adsorber particles to produce the ability that load has the film or the pan of particle.(90% sorbent material: 10%PTFE's particle closely combines in by weight) at the inert base of tetrafluoroethylene.The PTFE protofibril hinders the activity of particle never in any form.The extraction medium that EMPORE film manufacturing process produces is compared with the medium by the particle preparation of identical size that uses in conventional solid phase extractions (SPE) post or cylinder, can obtain finer and close, more uniform medium.

In a further preferred embodiment, solid phase (as, microporosity thermoplastic polymer carrier) have with connect by protofibril isolated, random dispersion, have inhomogeneous shape, each to etc. big a plurality of thermoplastic polymer particles microporosity structure that is feature.The particle each interval provides the network of micropore between them.Particle is connected to each other by the protofibril from each bombardment to adjacent particles.In particle or the protofibril one or both can be hydrophobic.Preferred this examples of material has higher surface area, as passing through mercury surface-area commercial measurement often up to 40 meters ²/ gram and the most about 5 micron pore size.

Such filamentary material can utilize the isolating optimization technique of induction phase to produce by relating to.This relates under the temperature that enough forms uniform mixture with immiscible liquid melt-mixing thermoplastic polymer, form goods with desired shape from the solution form, formed article is cooled off so that induce being separated of liquid and polymkeric substance, and polymer cure and the liquid of removing signal portion stay the microporosity polymeric matrix the most at last.This method and preferable material be at United States Patent (USP) 4,726, describes in detail among 989 (Mrozinski), 4,957,943 people such as () McAllister and 4,539,256 (Shipman).This material is called thermic phase separation membrane (TIPS film) and is particularly preferred.

Other suitable solid phase material is included in United States Patent (USP) 5,328, disclosed nonwoven material among 758 (people such as Markell).This material comprises and contains compression or merge the non-woven tablet of particulate (primitive fiber of preferred blowing) that it comprises the chromatographic grade particle of high adsorption efficiency.

Other suitable solid phase material comprises those that are called as HIPE Foams, and it is described in for example U.S. Patent Publication 2003/0011092 people such as () Tan." HIPE " or " High Internal Phase Emulsion " be meant comprise the active phase (being generally oil phase) of successive and not with the miscible discontinuous phase of oil phase or the emulsion of common external phase (being generally water), wherein immiscible phase accounts at least 74 volume % of emulsion.Many foam of polymers of being made by HIPE are generally relative perforate.This means that great majority or all adjacent holes of Kong Douyu communicate unblockedly.Have window between the Kong Zaikong of the foamy structure of this perforate basically, it is enough big usually, transfers to another hole from a hole to allow liquid in foamy structure.

Solid phase material can comprise the position of catching that is used to catch inhibitor.In this article, " catching the position " is meant that the group that is covalently attached on the solid phase material (as, functional group) or non-covalent (as, hydrophobicity) are incorporated into the molecule on the solid phase material.

Preferably, solid phase material comprises the functional group that catches inhibitor.For example, solid phase material can comprise chelation group.Here, " chelation group " for multiple-limb and can with atoms metal or ion (though inhibitor may by or may not remain on the solid phase material by chelating mechanism) form those of chelate complexes.The introducing of chelation group can realize by multiple technologies.For example, nonwoven material can hold with the functionalized globule of chelation group.Perhaps, the fiber of nonwoven material can directly carry out functionalized with chelation group.

The example of chelation group for example comprises-(CH ₂-C (O) OH) ₂, three (2-amino-ethyl) amine groups, iminodiacetic acid (salt) acid groups, nitrilotriacetic acid(NTA) group.Can chelation group be incorporated in the solid phase material by multiple technologies.They can be introduced into by the chemosynthesis of material.Perhaps, can with polymer-coated (as the coating of, figure) that comprise required chelation group to inert substrate (as, polycarbonate or polyethylene) on.If expectation can be duplicated substrate is little, be used to increase surface-area and strengthen and purify.Can also use oxygen plasma, electron beam or uv-radiation, heating or the pre-treatment of corona treatment method.Substrate can be used as for example mulch film on the bank of microfluidic device, or is laminated to mulch film.

The chelating solid phase material is commercially available and can be used as solid phase material in the present invention.For example, for certain embodiments of the present invention, preferably include the EMPORE film of chelation group such as iminodiethanoic acid (for the form of sodium salt).The particle of this film is at United States Patent (USP) 5,147, and is open and can be used as EMPORE Extraction Disks (47mm, No.2271 or 90mm are No.2371) available from 3M Company among 539 people such as () Hagen.For certain embodiments of the present invention, preferably include the EMPORE film of the ammonium derivatize of chelation group.For making pan be in ammonium form, its 0.1M ammonium acetate buffer with 50mL (pH 5.3) can be washed, wash with several reagent waters subsequently.

Other chelating examples of material includes but not limited to crosslinked polystyrene beads, and it can derive from Bio-Rad Laboratories, and (Hercules is under trade name CHELEX CA) for Inc.; Crosslinked agarose beads with three (2-amino-ethyl) amine, iminodiethanoic acid, nitrilotriacetic acid(NTA), polyamine and poly-imines; And available from Rohm and Haas (Philadelphia, trade name DUOLITE C-467 PA) and the chelating ion exchange resin under the DUOLITE GT73; AMBERLITE IRC-748, DIAION CR11, DUOLITE C647.

Usually, the desired concn density of the chelation group on the solid phase material be about 0.02 receive rub/square millimeter, though think that wide range of concentrations density is possible.

The capture material of other type comprises anion-exchange material, cation exchange material, activated carbon, anti-phase, positive, vinylbenzene-Vinylstyrene, aluminum oxide, silicon-dioxide, zirconium white and metallic colloid.The example of suitable anion-exchange material comprises strong anion exchanger such as quaternary ammonium, dimethylethanolamine, quaternary alkylamine, tri methyl benzyl ammonium and the dimethyl ethanol hexadecyldimethyl benzyl ammonium that is generally chloride form; With weak anionite, as polyamines.The example of suitable cation exchange material comprises strong cationite such as sulfonic acid, is generally sodium-salt form; With weak cationite such as carboxylic acid, be generally the form of acid.The example of suitable carbonaceous material comprises EMPORE carbon material, carbon pearl.Suitable anti-phase C8 and C18 examples of material comprise with octadecyl or octyl group carries out end capped silica bead and has C8 and the EMPORE material of C18 silica bead (deriving from 3M Co., St.Paul, the EMPORE material of MN).The positive examples of material comprises hydroxyl and dihydroxyl.

Also commercially available material can be carried out modification or be directly used in the method for the present invention.For example, can trade name LYSE AND GO (Pierce will be derived from, Rockford, IL), RELEASE-IT (CPG, NJ), GENE FIZZ (Eurobio, France), GENERELEASER (Bioventures Inc., Murfreesboro, TN) and BUGS NBEADS (GenPoint, Oslo, Norway) solid phase material, and Zymo globule (ZymoResearch, Orange, CA) and Dynal globule (Dynal, Oslo Norway) is attached in the method for the present invention as the solid-phase capture material, particularly is attached in the microfluidic device.

In some embodiment of this method, solid phase material comprises coating reagent.Preferred coatings reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.In some embodiment of this method, solid phase material comprises the tetrafluoroethylene fibril-matrix, be absorbed in the adsorptivity particle in the matrix and be applied to coating reagent on the solid phase material, and wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.In this article, phrase " is applied to the coating reagent on the solid phase material " and is meant the material at least a portion that is coated on solid phase material, as, be coated on the material at least a portion of fibril-matrix and/or adsorptivity particle.

The example of suitable tensio-active agent is listed below.

Suitable alkaline example comprises NaOH, KOH, LiOH, NH ₄OH, and primary amine, secondary amine or tertiary amine.

The example of suitable polyelectrolyte comprise polystyrolsulfon acid (as, poly-(4-Sodium styrene sulfonate) or PSSA), polyvinyl phosphonic acids, polyvinyl boric acid, polyvinylsulfonic acid, polyvinyl sulfuric acid, polystyrene phosphonic acids, polyacrylic acid, polymethyl acrylic acid, sulfonated lignin, carrageenin, heparin, chondroitin sulfate (chondritin sulfate) and salt or other derivative.

The example of the polymkeric substance barrier of suitable alternative infiltration comprises polymkeric substance such as acrylate, acrylamide, azlactone, polyvinyl alcohol, polymine, polysaccharide.This polymkeric substance can be various ways.They can be water miscible, water is expandable, water-insoluble, form of hydrogels etc.For example, the polymkeric substance barrier can be prepared as and make it play as the effect than macroparticle such as white corpuscle, nucleus, virus, bacterium and nucleic acid such as human gene group DNA and proteinic strainer.These surfaces can be by those skilled in the art by suitably selecting functional group, being suitable for separating according to size and/or electric charge by crosslinked etc. the transformation.This material is buied easily or can easily be prepared by those skilled in the art.Preferably, solid phase material is inexcessively washed away any tensio-active agent with tensio-active agent coating, though can wash other coating reagent off if desired.Usually, coating can several different methods be carried out, as dipping, roller coat, spraying etc.Usually before using, will be loaded with the solid phase material of coating reagent then at for example air drying.

Preferably, solid phase material is inexcessively washed away any tensio-active agent with tensio-active agent coating, though can wash other coating reagent off if desired.Usually, coating can several different methods be carried out, as dipping, roller coat, spraying etc.Usually before using, will be loaded with the solid phase material of coating reagent then at for example air drying.

What need especially is to scribble tensio-active agent, preferably scribble the solid phase material of nonionic surface active agent.This can realize according to the operation of mentioning in the embodiment part.Though do not plan to be limited by theory, think that the adding of tensio-active agent increases the wettability of solid phase material, its permission inhibitor penetrates in the solid phase material and with it and combines.

The coating reagent that is preferred for solid phase material is a group water solution, though with an organic solvent (alcohol etc.) if desired.The coating reagent heap(ed) capacity is should be enough high, makes the sample solid phase material that can drench.Yet it can not be too high, makes coating reagent itself by remarkable wash-out.Preferably, if coating reagent nucleic acid wash-out contains the coating reagent that is no more than about 2 weight % in the sample of wash-out.Usually, the concentration of coating solution in solution, can hang down the coating reagent that reaches 0.1 weight % and in solution up to the coating reagent of 10 weight %.

Tensio-active agent

Nonionic surface active agent.It is known that multiple suitable nonionic surface active agent is arranged, and it can be used as the coating on the optional solid phase material of cracking agent (above-mentioned discussion), eluent (following discussion) and/or conduct.They comprise for example polyoxyethylene surfactant, carboxylicesters tensio-active agent, carboxylic acid amide tensio-active agent etc.Commercially available nonionic surface active agent comprises the n-dodecane acyl sucrose; dodecyl-β-D-glycopyranoside; n-octyl-β-D-pyrans maltoside; n-octyl-β-D-sulfo-glycopyranoside; the n-dodecane acyl sucrose; positive decyl-β-D-pyrans maltoside; positive decyl-β-D-sulfo-maltoside; n-heptyl-β-D-glycopyranoside; n-heptyl-β-D-sulfo-glycopyranoside; n-hexyl-β-D-glycopyranoside; n-nonyl-β-D-glycopyranoside; positive capryloyl sucrose; n-octyl-β-D-glycopyranoside; cyclohexyl-n-hexyl-β-D-maltoside; cyclohexyl-N-methyl-β-D-maltoside; digitonin; with derive from trade name PLURONIC; TRITON; under the TWEEN those, and many other commercially available and that in Kirk Othmer Technical Encyclopedia, enumerate those.Its example is enumerated in following table 1.Preferred surfactants is a polyoxyethylene surfactant.Preferred tensio-active agent comprises octylphenoxy polyethoxy ethanol.

Table 1

The tensio-active agent trade(brand)name	Nonionic surface active agent	Supplier
			PLURONIC F127	The alcohol and/or the propenoxylated straight chain alcohol of the ethoxylation of modification	Sigma St.Louis，MO
TWEEN 20	Polyoxyethylene (20) mono laurate sorbitan ester	Sigma St.Louis，MO
			TRITON X-100	Uncle's octylphenoxy polyethoxy ethanol	Sigma St.Louis，MO
BRIJ 97	Polyoxyethylene (10) oleyl ether	Sigma St.Louis，MO
			IGEPAL CA-630	Octylphenoxy gathers (vinyloxy group) ethanol	Sigma St.Louis，MO
TOMADOL 1-7	Ethoxylated alcohol	Tomah Products Milton，WI
			Vitamin E TPGS	D-alpha-tocopherol cetomacrogol 1000	Eastman Kingsport，TN

Also suitable is the fluorinated nonionic type tensio-active agent of disclosed type in U.S. Patent Publication 2003/0139550 (people such as Savu) and 2003/0139549 (people such as Savu).Other non-ionic type fluorinated surfactant comprises (Wilmington, those under trade name ZONYL DE) available from DuPont.

Zwitterionics.Known multiple suitable zwitterionics, it can be used as coating on the solid phase material, as cracking agent and/or as eluent.They comprise for example alkyl amido betaine and amine oxide, alkyl betaine and amine oxide thereof, sultaine, hydroxyl sulfo betaine, both sexes glycinate, both sexes propionic ester, equilibrated both sexes many carboxyls glycinate and alkyl polyamino glycinate.Protein has charged or uncharged ability according to pH, and therefore at suitable pH, preferred pI as the bovine serum albumin or the chymotrypsinogen of modification, can be used as zwitterionics for the protein of about 8-9.The object lesson of zwitterionics is the courage amidopropyl dimethyl propylene ammonium sulphonate that derives under the trade name CHAPS of Sigma.Preferred tensio-active agent comprises N-dodecyl-N, N-dimethyl-3-ammonium-1-propane sulfonate.

Cats product.Known multiple suitable cats product, it can be used as cracking agent, eluent and/or as the coating on the solid phase material.They comprise for example quaternary ammonium salt, polyoxyethylene alkylamine and alkyl amine oxide.Usually, suitable quaternary ammonium salt comprises higher group of at least one molecular weight and the lower group of two or three molecular weight, they are connected in shared nitrogen-atoms with the generation positively charged ion, and wherein the negatively charged ion of balance electronic is selected from halogenide (bromide, muriate etc.), acetate moiety, nitrite anions and low alkyl group sulfonate radical (methanesulfonate etc.).The higher substituting group of molecular weight on the nitrogen often is to comprise about 10 senior alkyls to about 20 carbon atoms, the substituting group of lower molecular weight can be has 1 to the alkyl of about 4 carbon atoms such as the low alkyl group of methyl or ethyl, it can be substituted in some cases, as being replaced by hydroxyl.One or more substituting groups can comprise aryl moiety or can be replaced by aryl, as benzyl or phenyl.The possible substituting group with lower molecular weight also can comprise about 1 low alkyl group to about 4 carbon atoms, as methyl and ethyl; The substituted rudimentary poly-alkoxyl group part that has a hydroxyl end groups as the polyoxyethylene part, and has following general formula:

R(CH ₂CH ₂O) _(n-1)CH ₂CH ₂OH

Wherein R is (C1-C4) divalent alkyl that is incorporated on the nitrogen, and n represents about 1 to about 15 integer.Perhaps one or two this rudimentary poly-alkoxyl group with terminal hydroxyl can directly be incorporated into quaternary nitrogen, rather than is incorporated into nitrogen by aforementioned low alkyl group.The example that is used for useful quaternary ammonium halides tensio-active agent of the present invention includes but not limited to derive from methyl-two (2-hydroxyethyl) cocoyl ammonium chlorides or oleyl ammonium chloride (being respectively ETHOQUAD C/12 and O/12) and methyl polyoxyethylene (15) octadecyl ammonium chloride (ETHOQUAD 18/25) of Akzo Chemical Inc..

Anion surfactant.Known multiple suitable anion surfactant, it can be used as cracking agent, eluent and/or as the coating on the solid phase material.Spendable aniorfic surfactant comprises sulfonate and vitriol, as alkyl-sulphate, sulfated alkyl ether, alkylsulfonate, alkylether sulfonate, alkylbenzene sulfonate, alkylbenzene ether sulfate, alkyl sulfoacetate, secondary alkyl sulfonate, secondary alkyl sulfate etc.Have in these many can comprise the poly-alkoxylation group (as, ethylene oxide group and/or propylene oxide group, it can be the layout of random, orderly or block) and/or cationic counterion such as Na, K, Li, ammonium, protonated tertiary amine such as trolamine or quaternary ammonium group.Its example comprises: alkylether sulfonate, as available from Stepan Company, Northfield, the trade name POLYSTEP B12 of IL and the sodium lauryl ether sulphates under the B22, with available from Nikko Chemicals Co., Tokyo, the N-methyltaurine sodium under the trade name NIKKOL CMT30 of Japan; Available from Clariant Corp., Charlotte, the secondary alkyl sulfonate under the trade name HOSTAPUR SAS of NC, it is (C14-C17) Seconary Alkane Sulphonate Sodium (sulfonated); Methyl-2-sulfo group alkyl ester is as methyl-2-sulfo group (C12-C16) ester sodium with available from 2-sulfo group (C12-C16) the lipid acid disodium under the trade name ALPHASTE PC-48 of Stepan Company; As all available from the dodecyl sulfoacetic acid sodium (trade name LANTHANOL LAL) of Stepan Co. and alkyl sulfoacetate and the alkyl sulfo succinate under the MAKABATE LO 100 (trade name STEPANMILD SL3); And alkyl-sulphate, as available from the ammonium lauryl sulfate under the trade name STEPANOL AM of Stepan Co..

Another kind of useful anion surfactant comprises phosphoric acid salt, as alkylphosphonic, alkyl ether phosphate, aralkyl phosphoric acid salt and aralkyl ethers phosphoric acid salt.Have in these many can comprise poly-alkoxy base (as, ethylene oxide group and/or propylene oxide group, its can be random, in order or the layout of block).That its example comprises is single-, two-and three-(alkyl glycerylether)-o-phosphoric acid ester, typically refer to available from the trilaureth-4-phosphoric acid salt under the trade name HOSTAPHAT 340KL of Clariant Corp.; With available from Croda Inc., Parsipanny, PPG-5ceteth 10 phosphoric acid salt under the trade name CRODAPHOS SG of NJ; And alkyl and alkylamidoalkyl alkyl dialkylamine oxide compound.The example of amine oxide tensio-active agent comprises those (all available from the Stepan Co.) under trade name AMMONYX LO, LMDO and the CO, and it is laurylamide base propyl-dimethyl amine oxide and hexadecyl amine oxide.

Elution technique

Be used to keep the embodiment of the solid phase material of inhibitor for use, the more spissated zone of the sample of material that can use multiple eluent wash-out to comprise to contain nucleic acid (as, nucleus) and/or the nucleic acid that discharges.This eluent can comprise water (preferred not the water of qiagen rnase enzyme), damping fluid, tensio-active agent (it can be cationic, anionic, non-ionic type or amphoteric ionic surfactant) or highly basic.

Preferably, eluent is (that is, pH is greater than 7) of alkalescence.For some embodiment, the pH of eluent is at least 8.For some embodiment, the pH of eluent is for the highest by 10.For some embodiment, the pH of eluent is for the highest by 13.If the nucleic acid of wash-out is directly used in amplification procedure such as PCR, then eluent should be formulated as and makes that each component concentrations can inhibitory enzyme (as, Taq polysaccharase) or stop amplified reaction.

The example of suitable tensio-active agent comprises above-mentioned those, particularly, and for being called as those of SDS, TRITON X-100, TWEEN, fluorinated surfactant and PLURONICS.Tensio-active agent is provided in group water solution usually, though with an organic solvent (alcohol etc.) if desired.In the preferred eluent surfactant concentrations minimum based on the gross weight of eluent be 0.1 weight/volume % (w/v%).In the preferred eluent surfactant concentrations based on the gross weight of eluent for being not more than 1w/v%.Can randomly stablizer such as polyoxyethylene glycol be used with tensio-active agent.

The example of suitable elution buffer comprises TRIS-HCl, N-[2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid] (HEPES), the 3-[N-morpholino] propanesulfonic acid (MOPS), piperazine-N, N '-two [2-ethanesulfonic acid] (PIPES), the 2-[N-morpholino] ethyl sulfonic acid (MES), TRIS-EDTA (TE) damping fluid, Trisodium Citrate, ammonium acetate, carbonate and supercarbonate etc.

Preferably, in the eluent concentration of elution buffer minimum be 10 millis rub (mM).Preferably, surfactant concentrations is not more than 2 weight % in the eluent.

Usually, preferably use basic solution to realize containing the wash-out of the nucleic acid of the material of nucleic acid and/or release.Though do not plan to be bound by theory, think that and wash out-of-phase with water relatively, basic solution can improve the combination of inhibitor.Basic solution also promotes to contain the cracking of the material of nucleic acid.Preferably, the pH of basic solution is 8 to 13, more preferably 13.The example in the source of high pH comprises the aqueous solution of NaOH, KOH, LiOH, quaternary nitrogen based hybroxide, tertiary amine, secondary amine or primary amine etc.If basic solution is used for wash-out, usually it is neutralized with for example TRIS damping fluid in step subsequently, be ready for the sample of PCR with formation.

The use of basic solution can optionally destroy RNA, with permission DNA is analyzed.Otherwise, can in preparation, add rnase so that the RNA deactivation, subsequently with the hot deactivation of rnase.Similarly, can add deoxyribonuclease is used for optionally destroying DNA and allows RNA is analyzed; Yet, can with do not destroy RNA other lysis buffer (as, TE) be used for this method.Can also add ribonuclease inhibitor such as RNAsin to the preparation of the RNA preparation that is used for experiencing PCR in real time.

Wash-out at room temperature carries out usually, though higher temperature can produce higher yield.For example, if desired, the temperature of eluent can be up to 95 ℃.Wash-out carried out in 10 minutes usually, though preferred 1-3 minute elution time.

Device and test kit

The exemplary of multiple microfluidic device has been described in U.S. Patent Publication 2002/0047003 (on April 25th, 2003 is disclosed, people such as Bedingham).These common uses are furnished with the agent structure of incorporate microfluidic channel network therein.Aspect preferred, the agent structure of microfluidic device comprises the aggregate of two or more separate layers, described two or more separate layer forms microfluidic device of the present invention when suitably cooperating or combining, as comprises described passage and/or chamber herein.Usually, useful microfluidic device comprises top portion, bottom branch, internal portion, and wherein internal portion defines the passage and the chamber of device basically.Usually, the chamber comprises valve (as, valve partition) and is called the chamber that valve is arranged.

The particularly preferred device that is used for some embodiment of this paper is called control valve device, open in the applicant's that its title of submitting on December 12nd, 2003 is " Variable Valve Apparatus and Method " transferee's the U.S. Patent application co-pending 10/734,717.In this control valve device, valve arrangement allows to remove the selected part that is positioned at the specimen material in the treatment chamber (that is the treatment chamber that variable valve, is arranged).Removing by form opening in the valve partition in the predetermined position of selected part realizes.

The preferred valve partition is enough big, to allow the position according to the adjustment of features opening of specimen material in the treatment chamber.If rotary sample treatment unit after forming opening, then the material of the selected part of more close turning axle leaves treatment chamber by opening.The rest part of specimen material can not leave by opening, because its ratio open is farther apart from turning axle.

Can be in the valve partition in certain position according to one or more features of the specimen material that in treatment chamber, detects and form opening.The preferably treatment chamber comprises the detection window of the treatment chamber that light is imported into and/or spread out of.The feature of the specimen material that detects can comprise for example free surface of specimen material (capacity of specimen material in the characterization process chamber).Be in the valve partition at the radially outer selected distance of free surface and form opening, the ability of removing the specimen material of selected capacity from treatment chamber can be provided.

In some embodiments, might form opening by the select location place in one or more valve partitions and remove selected specimen material aliquot sample.Selected aliquot sample capacity can be measured according to the cross-sectional area of the treatment chamber between radial distance between the opening (measuring with respect to turning axle) and the opening.

Opening in the preferred valve partition forms under the situation that does not have the physics contact, as, by laser ablation, focused light heating etc.As a result, be preferably formed opening and do not penetrate the outermost layer of sample processing device, thereby limited the possibility of specimen material from the sample processing device seepage.

In one aspect, the present invention sample processing device (as, microfluidic device) uses in the valve treatment chamber is arranged, there is the valve treatment chamber to comprise treatment chamber with chamber enclosure, chamber enclosure is between the first and second relative major opposing sides of sample processing device, wherein treatment chamber occupies the process chamber area in the sample processing device, and wherein process chamber area have length and with the vertical width of length, and in addition wherein length greater than width.The treatment chamber that variable valve is housed also comprises the valve chamber that is positioned at process chamber area, valve chamber is between second major opposing side of chamber enclosure and sample processing device, wherein make valve chamber and treatment chamber isolation by the valve partition that valve chamber and treatment chamber are separated, and wherein the part of chamber enclosure between first major opposing side of valve partition and sample processing device.Detection window is positioned at process chamber area, and wherein detection window can see through selected electromagnetic energy, and this selected electromagnetic energy is introduced into and draws chamber enclosure.

In yet another aspect, the invention provides the method that permission is removed a part of sample from the treatment chamber selectivity that variable valve is housed.This method comprises provides aforesaid sample processing device (as, microfluidic device), sampling material in treatment chamber; Detect the feature of specimen material in the treatment chamber by detection window; Be in the valve partition at the select location along treatment chamber length and form opening, wherein Xuan Ding position is relevant with the specimen material feature of detection.This method also comprises by the opening that forms in the valve partition only to be transferred to a part of specimen material the valve chamber from treatment chamber.

The present invention also provides test kit, the teachings that it can comprise microfluidic device, cracking agent (tensio-active agent particularly, as nonionic surface active agent, form pure or in solution) and be used to make inhibitor and separate nucleic acid.

Other component that can comprise in the test kit of the present invention comprises conventional reagent, as washing lotion, coupling buffer, cancellation damping fluid, sealing damping fluid, elution buffer, or the like.Other assembly that can comprise in the test kit of the present invention comprises conventional equipment, as centrifugal post, tube core, 96 hole filter plates, needle-based strainer, collector unit, syringe, or the like.

Test kit generally includes wrapping material, and it is meant one or more physical structures that are used to hold the test kit content.Wrapping material can be constructed by known method, are preferred for the environment that does not contain pollutent that provides aseptic.Wrapping material can have the label that is used for visualizingre agent box content.In addition, test kit comprises and is used to indicate teachings how to use material in the test kit.As used in this article, term " packing " is meant solid substrate or material, as glass, plastics, paper, paper tinsel etc.

" teachings " generally includes actual expression, several different methods of the present invention is described, comprise cracking condition (as, the type of cracking agent and concentration), want blended reagent and sample the holding time of relative quantity, reagent/specimen mixture, temperature, buffer condition, or the like.

Illustrative methods

In preferred embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: the microfluidic device that comprises feed compartment, valve treatment chamber and mixing section are arranged is provided; The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided; Sedimenting reagent is provided; Sample is placed feed compartment; With sample transfer to the valve treatment chamber is arranged; Use sedimenting reagent in the valve treatment chamber is arranged, to form the sample concentration district, wherein the sample concentration district comprises that major part contains the material of nucleic acid, comprise the sample time enrichment region of at least a portion sedimenting reagent (preferred most of sedimenting reagent) and at least a portion inhibitor (randomly, can be before precipitation step for example water with sample dissociation) with formation; Start the valve have in the valve treatment chamber with at least a portion sample concentration district is transferred to mixing section and make basically enrichment region with inferior from differentiation from; The material cracking that will contain nucleic acid in mixing section is to discharge nucleic acid; Randomly regulate the pH of the sample of the nucleic acid that comprises release.Sedimenting reagent is for as discussed above.

The material that contains nucleic acid can be identical or different with the cell that contains inhibitor, though they are normally different.That is to say that the material that contains nucleic acid might be identical with the cell that contains inhibitor.For example, if sample is a buffy coat, the material that then contains nucleic acid can be white corpuscle, and it comprises nucleus and inhibitor simultaneously.If the cracking agent that uses (as, nonionic surface active agent) the leukocytic cytolemma of cracking rather than its interior contained nucleus, then inhibitor and complete nucleus are released together, and it also is considered to the material that contains nucleic acid of this paper definition.For some embodiment herein, through settled sample can comprise free (as, not in cell) inhibitor.

If expectation, before the material cracking that will contain nucleic acid, this method can comprise that water or damping fluid dilution are through isolating sample concentration district, the zone that randomly further concentrates the process dilution is to increase the concentration of nucleic acid substances, randomly separate through further spissated zone and randomly repeat this dilution and concentratedly subsequently reach separating process and reduce to the concentration that can not hinder amplification method with concentration inhibitor.

Perhaps, if expectation, before the material cracking that will contain nucleic acid, simultaneously or afterwards, this method can comprise the isolating sample concentration of process district transferred to and is used to contact solid phase material in the separate chamber, is attached to solid phase material preferentially to make at least a portion inhibitor; Wherein solid phase material comprise catch the position (as, huge legendary turtle is closed functional group), be applied to the coating reagent on the solid phase material or comprise both; Wherein coating reagent is selected from tensio-active agent, highly basic, polyelectrolyte, the permeable polymkeric substance barrier of selectivity.

With reference to figure 1, be applicable to that the preferred embodiment of the microfluidic device of these embodiments comprises feed compartment 50, optional mixing section 52, the amplified reaction chamber 62 that valve treatment chamber 54, the eluent chamber of choosing wantonly 58, waste chamber 60 is arranged and choose wantonly.These chambers are fluid communication each other, make sample be introduced in the feed compartment 50, can be transferred to mixing section 52 then, if or its do not exist and can directly transfer in the valve treatment chamber 54.

Can use preloaded (that is predeposition) in valve treatment chamber 54 is arranged or the sedimenting reagent that after adding sample, joins in the chamber having in the valve treatment chamber 54 sample concentration.In case sample and sedimenting reagent (as, the dextran aqueous solution) merge, they are mixed and permission generation sedimentation.There is the valve of valve treatment chamber 54 to be arranged so that usually and sample concentration district (it comprises that major part contains the material of nucleic acid) can be separated with sample time enrichment region (it often comprises most of sedimenting reagent and most of inhibitor) basically.Usually sample time enrichment region is transferred in the waste product chamber 60.The sample concentration district can directly transfer in the chamber standby, for example transfers in the Room 62, amplified reaction chamber.The cracking agent and the sample concentration district that storage can be referred to herein as in the eluent chamber 58 merge, and are used for further cracking.Perhaps, the sample concentration district can be transferred to the mixing section (not shown), be used for combining to discharge nucleic acid and/or to be used to regulate the pH of the sample of the nucleic acid that comprises release with cracking agent.

Other embodiments

As mentioned above, in damping fluid (particularly TRIS damping fluid), add sucrose and can help nuclear separation.Damping fluid also can comprise magnesium salts and tensio-active agent such as TRITON X-100.This also can be leukocytic cracking good medium is provided.In addition, in some cases, when the needs collecting cell is examined, particularly in microfluidic device, use nucleus storage damping fluid to come in handy.Nucleus storage damping fluid can comprise sucrose, magnesium salts, EDTA, dithiothreitol (DTT), 4-(2-amino-ethyl) benzene sulfonyl fluorine (AEBSF) and/or glycerine and allow nuclear stable state storage in for example damping fluid (as, TRIS damping fluid).

In some embodiment that relates to these methods of using microfluidic device, in the valve treatment chamber is arranged, form the sample concentration district and be included in the treatment chamber sample is centrifugal.Inferior enrichment region comprises sediment such as red corpuscle, and it is not transferred to Anywhere usually; But the more concentrated zone that will comprise nucleic acid is usually controlled (valved) with valve and is transferred in the other chamber of wherein it further being handled.

In some embodiment of described method, sample can be whole blood in this article.Then usually with separation of whole blood for each integral part and will comprise that leukocytic part (being commonly referred to buffy coat) is separated and cracking to discharge nucleus and/or nucleic acid.For example, in certain embodiments, this method can comprise with whole blood centrifugal (as, in the valve treatment chamber is arranged) to form plasma layer (often being the upper strata), red corpuscle layer (often being lower floor) and to comprise leukocytic interfacial layer, and remove the interfacial layer (that is buffy coat) of signal portion.The buffy coat experience is further handled.

In certain embodiments, can use ordinary method from the separation of whole blood buffy coat.Buffy coat can be used as the sample of described method herein then.

In certain embodiments, can use _ _ _ title submitted to for the U.S. Patent application of " METHODSFOR NUCLEIC ACID ISOLATION AND KITS USING SOLID PHASEMATERIAL " _ _ _ (Attorney Docket No.59073US003) in disclosed solid phase material remove inhibitor.

In some cases, can remove inhibitor by a series of concentrate/separate/optional dilution step, as _ _ _ title submitted to be " METHODS FOR NUCLEIC ACIDISOLATION AND KITS USING A MICROFLUIDIC DEVICE ANDCONCENTRATION STEP " U.S. Patent application _ _ _ disclosed in (Attorney DocketNo.59801US002).For example, when sample was blood, after RBC and sedimenting reagent settled, supernatant liquor (separated portions) comprised nucleic acid substances (in WBC), from hemolytic inhibitor of the generation of small portion RBC (because by water and cracked) and serum protein.This separated portions can through concentrating/separate/optional dilution step to be to reduce concentration that hemolytic RBC takes place (as, ferruginous inhibitor).

For communicable disease, have necessity the bacterium or the virus that derive from whole blood are analyzed.For example, in the situation of bacterium, white corpuscle may be to exist with bacterial cell bonded form.In microfluidic device, might use sedimenting reagent that erythrocyte sedimentation is come out, further isolating bacterial cell and white corpuscle before the cracking then.The concentrating part of this cell that contains nucleic acid (bacterium with leukocytic cell/nucleus) can further be transferred in the chamber, is used to remove inhibitor.Then, can be with for example bacteria cell cracking.

Can use heat to realize the cracking of bacterial cell, decide according to the type of bacterial cell.Perhaps, can use enzyme urge method (as, N,O-Diacetylmuramidase, mutanolysin) or chemical method carry out the cracking of bacterial cell.The preferred bacterium cell carries out cracking by alkaline lysis.

Blood plasma and serum have been represented and have been submitted the most of samples that comprise virus that are used for the molecule check to.After whole blood carried out classification, blood plasma or serum sample can be used for extracting virus (that is virus particle).For example, for from viral DNA isolation, might at first isolate red corpuscle by the use sinking agent.Then can isolating strong solution is centrifugal with concentrating virus or can use solid phase material or by being directly used in cleavage step subsequently herein after for example described a series of dilution/enrichment steps are removed inhibitor.

Solid phase material can absorbent solution, makes virus particle not pass through material.Then can be with little elution volume wash-out virus particle.By heating or by enzymatic or chemical process with virolysis (for example carrying out cracking) and be used for application such as the PCR or the PCR in real time in downstream by the use tensio-active agent.Need therein in the situation of viral RNA, may need ribonuclease inhibitor is joined in the solution, to stop the RNA degraded.

Therefore, except that above-mentioned solid phase material and sedimenting reagent, in multiple embodiments of the present invention can with the solid phase material of other type particularly globule be incorporated in the microfluidic device.For example, can be globule is functionalized with suitable group, be used to separate concrete cell, virus, bacterium, protein, nucleic acid, or the like.Can be by centrifugal and separating subsequently from the sample separation globule.Globule can be designed to have and be used for isolating appropriate density and size (nanometer is to micron).For example, in the situation that virus is caught, can before or after the inhibitor of from serum sample, removing small amount of residual, use the globule of the protein enclosure of identification virus to catch and make viral concentrating.

Can extract nucleic acid from isolating virus particle by cracking.Therefore, globule can be provided in the method that concentrates associated materials in the interior specific region of microfluidic device, also allows to wash uncorrelated material and wash-out associated materials from the particle of catching.

The example of this globule includes but not limited to crosslinked polystyrene beads, it can derive from Bio-Rad Laboratories, Inc. (Hercules, CA) under the trade name CHELEX, has the crosslinked agarose beads of three (2-amino-ethyl) amine, iminodiethanoic acid, nitrilotriacetic acid(NTA), polyamine and poly-imines; And available from Rohm and Haas (Philadelphia, trade name DUOLITE C-467 PA) and the chelating ion exchange resin under the DUOLITE GT73; AMBERLITE IRC-748, DIAION CR11, DUOLITE C647.These globules also are suitable as aforesaid solid phase material.

Other example of globule comprises (the Eurobio available from trade name GENE FIZZ, France), GENE RELEASER (Bioventures Inc., Murfreesboro, TN) and BUGS NBEADS (GenPoint, Oslo, Norway) under those, and the Zymo globule (ZymoResearch, Orange is CA) with DYNAL globule (Dynal, Oslo, Norway).

Other material also can be used for pathogenic agent and catches.For example, polymeric coating also can be used to separate specific cell, virus, bacterium, protein, nucleic acid etc. in certain embodiments of the invention.These polymeric coatings can be sprayed on the mulch film of microfluidic device for example by direct jet.

Can be covalently bound on little bead surface and virus particle is captured on the globule by making antibody.Antibody can be used for resisting the coat protein of virus.For example, the DYNAL globule can be used for covalently bound antibody.Perhaps, synthetic polymer such as anion exchange polymer can be used for the concentrating virus particle.(Biotech Support Group, East Brunswick NJ) can be used for being coated with on globule or the select location in microfluidic device as polymeric coating, with the concentrating virus particle for commercially available resin such as viraffinity.(GenPoint, Oslo Norway) can be used for for example extraction of bacterium to BUGS N BEADS.Here, these globules can be used for catching bacterium such as staphylococcus (Staphylococcus), suis (Streptococcus), intestinal bacteria (E coli), Salmonellas (Salmonella) and Clamydia elementary body.

Therefore, in one embodiment of the invention, when sample comprises virus particle or other pathogenic agent when (as, bacterium), microfluidic device can comprise that virus catches the solid phase material of globule or other pathogenic agent capture material form.More specifically, in a kind of situation, globule can only be used for concentrating of virus or bacterium, for example subsequently globule is separated in another chamber, and finishes with the cracking of virus or bacterium.In another kind of situation, globule can be used for concentrating of virus or bacterium, and cracking and capture nucleic acid on identical globule with the globule dilution, concentrate globule subsequently, separate globule, and repeated this process repeatedly before the nucleic acid of elute captured.

If the downstream application of nucleic acid is to make its experience amplification procedure such as PCR, all reagent that then are preferred for this method all compatible with this process (as, PCR is compatible).In addition, the adding of PCR promotor (facilitators) comes in handy, especially for diagnostic purpose.In addition, the material heating that will increase before amplification may be favourable.

Do not remove fully therein in the embodiment of inhibitor, can add damping fluid, enzyme and PCR promotor, they help the amplification procedure in the presence of inhibitor.For example, can use the enzyme that is different from the Taq polysaccharase that inhibitor is had more tolerance,, thereby provide huge benefit for pcr amplification as rTth.Can add bovine serum albumin, trimethyl-glycine, protease inhibitor, ox transferrin etc., known they can further help amplification procedure.Perhaps, (EMDBiosciences, Darmstadt Germany), are used for from directly amplification and need not large-scale purifying of whole blood can to use commercially available product such as Novagen ' s Blood Direct PCR Buffer test kit.

Further specify objects and advantages of the present invention by following examples, but the concrete material narrated in these embodiments and amount thereof and other condition and details should be interpreted as and exceedingly limit the present invention.

Embodiment

The preparation of solid phase material: form the ammonia form without TRITON X-100

(3M, St.Paul MN) place the glass filter support with 3M No.2271 EMPORE Extraction Chelating Disk.According to the method for printing on the package insert extraction dish is converted into the ammonia form.

Dish is placed bottle and be immersed in 1%TRITON X-100 (Sigma-Aldrich, St.Louis, MO) in the solution (the TRITON X-100 of 0.1 gram (g) is in 10mL water), at Thermolyne Vari-Mix Model M48725 Rocker (Barnstead/Thermolyne, Dubuque IA) goes up mixing about 6-8 hour.Dish is placed on the glass filter support by applying about 20 minutes (min) dryings of vacuum, in room temperature (about 21 ℃) dried overnight down, note not washing and rinsing then.

Embodiment 1: obtain the method for DNA sample from using the settled white corpuscle that derives from whole blood of dextran

Remove leucocyte-removing by the differential sedimentation in dextran/salts solution from whole blood according to method 1 (preparing white corpuscle-National ReferralLaboratory for Lysosomal by the dextran sedimentation, Peroxisomal and Related Genetic Disorders).The pure TRITON X-100 of one (1) μ L is joined in the white corpuscle of two (2) μ L.With solution vortex and in Eppendorf Model 5415D whizzer centrifugal about 1 minute momently at 400rcf.Three (3) μ L samples are placed on the chelating membrane of preparation as mentioned above.Allow material on film dry about 2-5 minute.The 0.077M NaOH that adds ten three (13) μ L to chelating membrane.If solution is spumescence, with its centrifugal 1 minute at 4,000 rev/mins (rpm).Solution after mixing 2-3 time up and down and mixing, is removed the transfer pipet end.Remove the aliquot sample of 2 μ L and join among the 40mMTRIS-Cl (pH 7.4) of 10 μ L.

Embodiment 2A: inhibitor/DNA is to the influence of PCR: the concentration that changes inhibitor under fixed DNA concentration

Before the pure human gene group DNA of admixture, make the serial dilution thing of inhibitor, so that the research inhibitor is to the influence of PCR.In the human gene group DNA of 15 nanograms/microlitre (ng/ μ L) of 10 μ L, add the different Mix I of 1 μ L (pure or its dilution) (sample 2-does not add inhibitor, and 2D-is pure, 2E-1: 10,2F-1: 30,2G-1: 100,300) and vortex 2H-1:.Two (2) the μ L aliquot sample that obtain each sample are carried out the PCR of 20 μ L.The result is as shown in table 2.

Mix I: the pure TRITON X-100 that in the whole blood of 100 (100) μ L, adds 1 μ L.Solution was cultivated about 5 minutes down in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it is transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer centrifugal about 10 minutes at 400rcf.Obtain about 80 μ L from the top of Eppendorf tube and be appointed as Mix I.

Embodiment 2B: inhibitor/DNA is to the influence of PCR: change DNA concentration under the fixed inhibitor concentration

The Mix I (aforesaid) that in the human gene group DNA of 10 μ L, adds the dilution in 1: 3 of 1 μ L.The DNA concentration of research is as follows: sample 2J-15ng/ μ L, 2K-7.5ng/ μ L, 2L-3.75ng/ μ L, 2M-1.5ng/ μ L.The aliquot sample that obtains two (2) μ L from each sample is used for the PCR of 20 μ L.The result is as shown in table 2.

Embodiment 2C: inhibitor/DNA is to the influence of PCR: the DNA that does not add inhibitor

The water that adds 1 μ L in each DNA sample replaces the following sample of inhibitor preparation: sample 2N-15ng/ μ L, 2P-7.5ng/ μ L, 2Q-3.75ng/ μ L, 2R-1.5ng/ μ L.The aliquot sample that obtains two (2) μ L from each sample is used for the PCR of 20 μ L.The result is as shown in table 2.

Table 2

Sample number into spectrum	Ct (duplicate sample)	Sample number into spectrum	Ct (duplicate sample)
				2	19.10 19.06	2K	29.16 30.22
2D	13.94 29.50	2L	30.47 29.96
				2E	27.39 26.22	2M	28.43 26.16
2F	21.44 20.66	2N	20.05 19.80
				2G	19.90 19.30	2P	20.74 20.54
2H	19.90 20.08	2Q	21.95 21.88
				2J	28.45 28.61	2R	22.67 23.10

The result

Table 3 reported according in the QuantiTech SYBR Green PCR Handbook 10-12 page or leaf about preparing the guidance of 10 μ L PCR samples (2 μ L samples in 10 μ L SYBR Green Master Mix, the beta-actin of 4 μ L, the water of 4 μ L), at ABI 7700 QPCRMachine (Applera, Foster City, the result of the embodiment 1-2 that obtains on CA).In these experiments, do not amplify no template contrast (NTC).Horizon 11-14 ElectrophoresisMachine (Gibco BRL, Gaithersburg, MD) go up operation 1% sepharose (bands of a spectrum brightness-+fuzzy, +++bright).Under 405nm at SpectraMax Plus ³⁸⁴(Molecular Devices Corporation, Sunnyvale CA.) carry out spectral measurement on the spectrophotometer.Two, three of each sample or four numeric representations are duplicate, in triplicate or quadruplicate.

Table 3

Sample	Ct	Bands of a spectrum	405nm (mean value)
				1.5ng/ μ L human gene group DNA in 0.1M NaOH/40mM TRIS-HCl damping fluid	16.92 20.67	+++ +++	-
1.5ng/ μ L human gene group DNA in water	19.01 18.67	+++ +++	0
				1.5ng/ μ L human gene group DNA in water	16.18 16.28	+++ +++	-
Embodiment 1	22.03	+++	-
				The Mix I of embodiment 2A and 2B dilution in 1: 36	-	-	2.63
The Mix I of embodiment 2A and 2B dilution in 1: 360	-	-	0.38
				The Mix I of embodiment 2A and 2B dilution in 1: 3600	-	-	0.036
The Mix I of embodiment 2A and 2B dilution in 1: 36000	-	-	0

Multiple change of the present invention and variation will become apparent to those skilled in the art that and do not depart from the scope of the present invention.Should be appreciated that the present invention is not subjected to exemplary that this paper proposes and the excessive restriction of embodiment, and this embodiment and embodiment exist as an example just, scope of the present invention is only by the following claim restriction that proposes herein.

Claims (13)

1. from the method for sample separation nucleic acid, this method comprises:

The microfluidic device that comprises feed compartment, valve treatment chamber and mixing section are arranged is provided;

Provide and comprise the material that contains nucleic acid and the sample of inhibitor;

Sedimenting reagent is provided;

Sample is placed feed compartment;

With sample transfer to the valve treatment chamber is arranged;

Use sedimenting reagent to form the sample concentration district in the valve treatment chamber is arranged, wherein the sample concentration district comprises that major part contains the material of nucleic acid, and sample time enrichment region comprises at least a portion sedimenting reagent and at least a portion inhibitor;

Start the valve have in the valve treatment chamber at least a portion sample concentration district transferred to mixing section and to make at least a portion sample concentration district and the inferior enrichment region of sample separates;

The material cracking that will contain nucleic acid in mixing section under the condition of optionally heating is to discharge nucleic acid; With

Optional adjusting comprises the pH of sample of the nucleic acid of release.

2. from the method for sample separation nucleic acid, this method comprises:

The microfluidic device that comprises feed compartment, valve treatment chamber and mixing section are arranged is provided;

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Sedimenting reagent is provided;

Sample is placed feed compartment;

With sample transfer to the valve treatment chamber is arranged;

Use sedimenting reagent to form the sample concentration district in the valve treatment chamber is arranged, wherein the sample concentration district comprises that major part contains the material of nucleic acid, and sample time enrichment region comprises at least a portion sedimenting reagent and at least a portion inhibitor;

Start the valve have in the valve treatment chamber at least a portion sample concentration district transferred to mixing section and to make at least a portion sample concentration district and the inferior enrichment region of sample separates;

The material cracking that will contain nucleic acid in mixing section is to discharge nucleic acid; With

Optional adjusting comprises the pH of sample of the nucleic acid of release.

3. the method for claim 2, wherein sample is a blood.

4. the method for claim 2, the material that wherein contains nucleic acid comprises nucleus.

5. the method for claim 2, wherein time enrichment region comprises most of sedimenting reagent.

6. the method for claim 2, wherein sample is a tissue extract.

7. the method for claim 2, its sample transfer that comprises the nucleic acid that will comprise release in addition is to the amplified reaction chamber.

8. the method for claim 7, it comprises the nucleic acid experience amplification procedure that makes release in addition.

9. the method for claim 2 wherein forms the sample concentration district and comprises and carry out centrifugal to the sample in the treatment chamber in the valve treatment chamber is arranged.

10. the method for claim 2, wherein before the material cracking that will contain nucleic acid, this method comprises that water or damping fluid dilution are through isolating sample concentration district, the zone that randomly further concentrates the process dilution is to increase the concentration of nucleic acid substances, randomly separate through further spissated zone, randomly repeat this dilution and concentrate subsequently to reach separating process, reduce to the concentration that can not hinder amplification method with concentration inhibitor.

11. the method for claim 2, wherein before the material cracking that will contain nucleic acid, simultaneously or afterwards, this method comprises that the isolating sample concentration of process district is transferred to the separate chamber is used to contact solid phase material, makes at least a portion inhibitor preferentially be attached to solid phase material; Wherein solid phase material comprises and catches the position, is applied to the coating reagent on the solid phase material or comprises both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.

12. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

Sedimenting reagent;

Microfluidic device, it comprises feed compartment, valve treatment chamber and mixing section is arranged; With

Teachings is used for according to the method for claim 1 sample dissociation being separated with at least a portion inhibitor with the material that makes major part contain nucleic acid.

13. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

Sedimenting reagent;

Microfluidic device, it comprises feed compartment, valve treatment chamber and mixing section is arranged; With

Teachings is used for according to the method for claim 2 sample dissociation being separated with at least a portion inhibitor with the material that makes major part contain nucleic acid.