CN101268189A - Method for isolating nucleic acids - Google Patents

Abstract

Described herein is a method in which genomic nucleic acid of a cell can be separated from nucleic acid having a molecular weight that is lower than the molecular weight of the genomic nucleic acid (e.g., plasmid DNA) of the cell directly from a cell growth culture. Also described herein, a method in which genomic nucleic acid can be separated from nucleic acid having a molecular weight that is lower than the molecular weight of the genomic nucleic acid in a cell lysate without the need to prepare a cleared lysate. The present invention is also directed to a method of isolating genomic nucleic acid (e.g., RNA, DNA) of a cell or an organism.

Description

The method of isolating nucleic acid

Related application

The application's case is the U. S. application case the 11/231st of application on September 20th, 2005, No. 363 the application case that continues, it is the U. S. application case the 10/406th of application on April 2nd, 2003, No. 141 part continue application case and for the international application case PCT/US2004/009960 number (it specifies the U.S., apply on April 1st, 2004, and open with English) the part application case that continues.Whole teachings of above-mentioned application case are to be incorporated herein by reference.

Background technology

Many molecular biology of considering transfection, transduction or the microinjection of mammalian cell are used (such as capillary electrophoresis, nucleotide sequencing, reverse transcription clone and gene therapy scheme) needs to separate the high-quality nucleic acid preparation.

The appearance that high request molecular biology is used has increased for the high yield that can produce the high-quality nucleic acid preparation and preferably has been easy to the demand of the purification scheme of automatization.Although the appearance of technical progress recently and robot technology has promoted the automatization of sequencing reaction and gel read step, output still is subjected to being easy to the restriction of operability of the nucleic acid purification method of automatization.

Summary of the invention

As described herein, the applicant provides a kind of method, wherein cell or organic genomic nucleic acids can be directly molecular weight is lower than the genomic nucleic acids molecular weight from cell or organism growth culture and cell or organism nucleic acid (for example plasmid DNA) separate.In addition, the applicant provides a kind of method, and wherein genomic nucleic acids can be lower than the separate nucleic acid of genomic nucleic acids molecular weight with molecular weight in cell or organism lysate, and need not preparation clarification lysate (lysate).

Tradition alkalescence dissolving (lysis) needs following steps: concentrated or granulation (pelleting) is diluted in the cell in the growth medium; Centrifugal or vortex; With alkaline washing agent dissolved cell; Vibration and/or stirring and dissolving product; Add neutralization buffer; Filter and/or handle sample to remove floss; Add solid phase carrier; And add binding buffer liquid.Usually need following other purification step to remove washing agent and salt: to add solid phase carrier and add binding buffer liquid.

The invention has the advantages that the simplifying procedures of separate nucleic acid that it allows molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the genomic nucleic acids molecular weight.By solid phase carrier being provided and making cell or organism dissolving and cell or organic nucleic acid be deposited in the reagent (nucleic acid precipitation agent) on the described solid phase carrier, can be from purifying from removing one or more step the standard purification method of complete or full cell or organic nucleic acid.In addition, by solid phase carrier being provided and making cell or organic nucleic acid be deposited in the reagent on the described solid phase carrier, can be from purifying from removing one or more step the standard purification method of the nucleic acid of cell or organism lysate.The order that solid phase carrier and reagent and cell, organism, cell lysates or organism lysate merge is not crucial.Solid phase carrier and make cell or organic nucleic acid be deposited on the described solid phase carrier and/or cell or organism dissolved reagent can be successively (for example, with independent component forms; In two steps) or simultaneously (for example, with the one-component form; In a step) merge with cell, organism, cell lysates or organism lysate.When solid phase carrier and reagent are merged into one-component, described herein method allows single agents is added in cell or organism or cell or the organic culture, then cultivate, separate (one or more) solid phase carrier and selective elution and be lower than the separating of nucleic acid of genomic nucleic acids molecular weight to realize cell or organic genomic nucleic acids and cell or organic molecular weight.Method of the present invention does not need the pH value to regulate.The number of steps of the minimizing that is provided by described reagent and method herein can be simplified the automatic operation of the nucleic acid purification method of cell, organism, cell lysates or organism lysate.

Therefore, the present invention relates to a kind of nucleic acid (plasmid DNA for example that molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the genomic nucleic acids molecular weight, episome DNA, Mitochondrial DNA, organelle DNA, viral DNA) isolating method, it comprises and merges i) solid phase carrier (for example magnetic particle), on its surface in conjunction with the functional group of reversible bind nucleic acid (carboxyl for example, amido), ii) cell or organism and iii) reagent, wherein said reagent makes cell or organism dissolving and cell or organic nucleic acid be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under cell or organism generation dissolving and cell or organic nucleic acid and the reversible bonded condition of solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic nucleic acid.Solid phase carrier separated with combination and with make molecular weight be lower than the nucleic acid of genomic nucleic acids to contact from the elution buffer (for example water) of solid phase carrier wash-out (selective elution).Genomic nucleic acids keeps combining with solid phase carrier, thereby makes molecular weight in cell or organic genomic nucleic acids and cell or the organism be lower than the separate nucleic acid of genomic nucleic acids molecular weight.

The present invention also relates to a kind of method of the plasmid separate nucleic acid with cell or organic genomic nucleic acids and cell, it comprises and merges i) magnetic particle, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism and iii) reagent is (for example, alcohol such as ethanol, Virahol, polyalkylene glycol), wherein said reagent makes cell or organism dissolving and cell or organic nucleic acid be deposited on the magnetic particle, thereby produces combination.Described combination is remained under cell or organism generation dissolving and cell or organic nucleic acid and the reversible bonded condition of magnetic particle, thereby produce the magnetic particle that has with magnetic particle bonded cell or organic nucleic acid.To have and separate with combination with the magnetic particle of magnetic particle bonded cell or organic nucleic acid and contact with the elution buffer that makes plasmid nucleic acid from the magnetic particle wash-out.Genomic nucleic acids keeps combining with solid phase carrier, thereby makes cell or organic genomic nucleic acids and cell or organic plasmid separate nucleic acid.

The present invention also relates to a kind of isolating method of nucleic acid (for example plasmid nucleic acid) that molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the genomic nucleic acids molecular weight, it comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism lysate and iii) reagent, wherein said reagent makes the nucleic acid of cell or organism lysate be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under the nucleic acid and the reversible bonded condition of solid phase carrier of cell or organism lysate, thereby produce the solid phase carrier have with the nucleic acid of solid phase carrier bonded cell.Solid phase carrier separated with combination and contact with the elution buffer that makes nucleic acid that molecular weight is lower than genomic nucleic acids from the solid phase carrier wash-out.Genomic nucleic acids keeps combining with solid phase carrier, thereby molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the separate nucleic acid of genomic nucleic acids molecular weight.

In the methods of the invention, can use any appropriate methodology (for example magnetic means, centrifugal) that genomic nucleic acids institute bonded solid phase carrier is separated from the elutriant that comprises molecular weight and be lower than the nucleic acid of genomic nucleic acids molecular weight.Solid phase carrier can be contacted with the suitable elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out subsequently.

(for example the present invention also relates to a kind of isolated cell, prokaryotic cell prokaryocyte, such as the eukaryotic cell of mammalian cell) or organism (pathogenic agent for example, such as virus, bacterium, parasite, fungi) the method for genomic nucleic acids (for example RNA, DNA), it comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism and iii) reagent, wherein said reagent makes cell or organism dissolving and cell or organic nucleic acid be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under cell or organism generation dissolving and cell or organic genomic nucleic acids and the reversible bonded condition of solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic genomic nucleic acids.Subsequently solid phase carrier is separated with combination, thus isolated cell or organic genomic nucleic acids.Solid phase carrier can be contacted with the elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out.

In a specific implementations, the present invention relates to the method for a kind of isolated cell or organic genomic nucleic acids, it comprises cell or organism and makes cell or organism dissolved reagent merge, thereby produces first combination; And described first combination is remained under cell or the organism dissolved condition, thereby produce lysate.Merge lysate and binding buffer liquid, described binding buffer liquid comprises solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid; And reagent, it makes cell or organic nucleic acid be deposited on the solid phase carrier, thereby produces second combination.Described second combination is remained under the reversible bonded condition of cell or organic genomic nucleic acids and solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic genomic nucleic acids.Subsequently solid phase carrier is separated from second combination, thus isolated cell or organic genomic nucleic acids.Described method can further comprise makes solid phase carrier contact with the elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out.

The present invention also relates to be applicable to the test kit in the described method herein.In one embodiment, described test kit comprises dissolving damping fluid, binding buffer liquid, the reagent that removes impurity and lavation buffer solution.Described dissolving damping fluid can comprise sodium lauryl sulphate, triton x-100 (Triton X-100), ethylenediamine tetraacetic acid (EDTA) (EDTA) and Tris-HCl.Binding buffer liquid can comprise magnetic particle, polyoxyethylene glycol and sodium iodide.The reagent that removes impurity can comprise the reagent (for example deoxyribonuclease (DNase)) of the reagent (such as Proteinase K) of digesting protein, dna digestion and/or the reagent (for example rnase (RNase)) of digestion RNA.Lavation buffer solution can comprise polyoxyethylene glycol and urea.In a specific implementations, test kit comprises i) the dissolving damping fluid, it comprises sodium lauryl sulphate, Triton X-100, EDTA and Tris; Ii) binding buffer liquid, it comprises magnetic particle, polyoxyethylene glycol and sodium iodide; Iii) Proteinase K; Iv) lavation buffer solution, it comprises polyoxyethylene glycol and urea.

Description of drawings

The scheme explanation of Fig. 1 for having in the genomic nucleic acids of cell and the cell than the more low-molecular-weight separate nucleic acid of genomic nucleic acids;

Fig. 2 is 96 hole sepharoses, and plasmid separates in the intestinal bacteria of its showed cell (E.coli) genomic nucleic acids and the cell;

Fig. 3 is by the Phred 20 (redness) of reading generation and the column diagram of Phred 30 (black) base; Y-axis is a number of readings per taken, and X-axis is that increment is Phred 20 combinations of 50bp;

Fig. 4 shows two parts of gDNA by the preparation of 50 μ l horse blood;

Fig. 5 shows that 8 PicoGreen by the gDNA sample of horse blood preparation analyze;

Fig. 6 shows by the grads PCR of the gDNA of horse blood preparation (using the Y3B19 mark of expection amplicon size as 225bp);

Fig. 7 is the agarose electrophoresis gel (e-gel) from the genomic dna of horse separation of whole blood;

Fig. 8 is the agarose e-gel from the genomic dna of pig separation of whole blood;

Fig. 9 is for the use dissolving with in conjunction with the agarose e-gel of the method that takes place with two steps from the genomic dna of people's separation of whole blood;

Figure 10 is the agarose e-gel from the isolating total RNA of mammalian cell that cultivates;

Figure 11 shows the capillary electrophoresis result from the isolating RNA of solid tissue;

Figure 12 shows from the capillary electrophoresis result and the agarose e-gel of the RNA sample of separation of whole blood;

Figure 13 shows that using gargles and collects agarose e-gel and the PCR result from the genomic dna of cheek cellular segregation;

Figure 14 shows agarose e-gel and the PCR result of use wiping collection from the genomic dna of cheek cellular segregation;

Figure 15 is presented at and separates in the plasma sample and detection hepatitis B virus (hepatitis B virus; HBV) the PCR result of genomic dna;

Figure 16 shows from the agarose e-gel and the PCR result of the genomic dna of paraffin embedding sample separation.

Embodiment

This patent or application document contain at least one and use chromatic graphic.Government bodies will provide the copy with colored graphic this patent or the open case of patent application after request and payment essential cost.

As described herein, the invention provides cell or the isolating method of organic nucleic acid (for example plasmid DNA) of using the minimal steps number cell and/or organic genomic nucleic acids and molecular weight can be lower than the genomic nucleic acids molecular weight.Directly pair cell or organism culture separate, and need not granulation cell or organism.In addition, described herein method is pair cell or the enforcement of organism lysate directly, and need not to use the lysate of traditional method (for example centrifugal, chemical treatment) clarification genomic nucleic acids.Nucleic acid at cell is mainly in the specific implementations of endogenous nucleic acid (for example DNA, RNA), the invention provides cell or organic endogenous nucleic acid (for example, genomic nucleic acids (for example DNA, RNA), mitochondrial nucleic acid, mitochondrial RNA(mt RNA), transfer RNA (tRNA), microRNA, messenger RNA(mRNA)) and cell or the isolating method of organism.Described method also can be used for cell or organic various endogenous nucleic acid (for example, interior source DNA being separated with endogenous RNA) separated from one another.

The invention provides the method and the reagent of isolating nucleic acid.Described herein reagent can be used for molecular weight in (one or more) cell or organic genomic nucleic acids and cell or the organism is lower than the separate nucleic acid of genomic nucleic acids molecular weight, it is by cell or organism are carried out with solid phase carrier and reagent merging, described reagent makes the dissolving of cell or organism, and cell or organic nucleic acid are deposited on the solid phase carrier.Perhaps, described herein reagent can be used for molecular weight in the genomic nucleic acids of cell or organism lysate and cell or the organism lysate is lower than the separate nucleic acid of genomic nucleic acids molecular weight, it is to be undertaken by cell or organism lysate are merged with solid phase carrier and reagent, and described reagent makes the nucleic acid of cell or organism lysate be deposited on the solid phase carrier.Subsequently molecular weight in cell or the organism is lower than the nucleic acid of genomic nucleic acids molecular weight from the solid phase carrier selective elution.In another embodiment, described herein reagent can be used for separating (one or more) cell or organic genomic nucleic acids, it is to be undertaken by cell or organism are merged with solid phase carrier and reagent, and described reagent makes cell or organism dissolving and cell or organic genomic nucleic acids be deposited on the solid phase carrier.

As described herein, method comprises the nucleic acid of cell, organism, cell lysates or organism lysate with to have functional group's bag non-specific and reversibly combine by the solid phase carrier (for example magnetic particle) on surface (for example the carboxyl bag is by the surface).Can for example to attract magnetic particle particulate be separated from supernatant liquor subsequently by applying magnetic field.Removable subsequently surplus solution (for example supernatant liquor) stays the particulate with bind nucleic acid.In case separate from supernatant liquor, the elution buffer that just can make molecular weight in particulate and selective elution cell or the organism be lower than the nucleic acid of genomic nucleic acids molecular weight contacts.Therefore, produce contain not bind nucleic acid (cell or organic nucleic acid, it has the molecular weight lower than the molecular weight of cell or organic genomic nucleic acids) and cell or organic genomic nucleic acids still with the elution buffer of its bonded magnetic particle.The nucleic acid that the concentration that is used for elution buffer that wash-out cell or organism molecular weight be lower than the nucleic acid of genomic nucleic acids molecular weight and is the nucleic acid precipitation reagent is lower than the genomic nucleic acids molecular weight at molecular weight is incorporated into the solution below the scope required on the magnetic particle.Basically all nucleic acid are in the embodiment of genomic nucleic acids in cell or organism, subsequently with particulate with the elution buffer of genomic nucleic acids from the particulate wash-out contacted.In one embodiment, eluent is a water.In addition, sucrose (20%) and methane amide (100%) solution can be used for wash-out nucleic acid.When using the elution buffer (for example water) of low ionic strength, in 30 seconds or shorter time the wash-out of nucleic acid from particulate takes place.In case bind nucleic acid through wash-out, just can separate magnetic particle from the elution buffer that contains through wash-out nucleic acid.Preferably magnetic particle is separated from elution buffer by magnetic means (magnetic means).Known other method of those skilled in the art can be used for magnetic particle is separated with supernatant liquor.For instance, can use filtration or centrifugal.

In one embodiment, in case will be still with cell or organic genomic nucleic acids bonded magnetic particle from (for example containing not bind nucleic acid, cell or organic nucleic acid, it has than cell or the low molecular weight of organic genomic nucleic acids molecular weight) elution buffer shift out, also can make described magnetic particle contact with suitable elution buffer with cell or organic genomic nucleic acids from the magnetic particle wash-out.The genomic nucleic acids that shifts out can be used for (for example) agarose gel analysis, pcr amplification, restriction enzyme digestion, diagnosis and/or treatment analysis, people's identity telling test, film hybridization (for example south and spot slit engram hybridization (Southern and dot/slot blots)) and AFLP, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), little satellite and mononucleotide polymorphic (SNP) and analyzes (for example, being used for genetic typing, fingerprint recognition etc.).

The starting soln of high-quality nucleic acid preparation and multicomponent and complicacy is separated into the basic technology in the molecular biology.Because described reagent and method herein, nowadays exist genomic nucleic acids and molecular weight are lower than separate nucleic acid rapid of genomic nucleic acids and are easy to automated method.Needing can be used in the molecular biology application of high-quality nucleic acid by institute's isolating nucleic acid of revealing method, preparation such as the dna sequencing template, microinjection, the transfection of mammalian cell or conversion, the external of RNAi hair clip synthesized, the reverse transcription clone, the cDNA library construction, pcr amplification and gene therapy study, and having more not other application of severity specification of quality, it includes, but is not limited to transform, restriction endonuclease or microarray analysis, selectivity RNA precipitation, external (in vitro) transposition, the separation of polynary pcr amplification product, the preparation of dna probe and primer and stripper plate scheme.

Described herein reagent and method can (comprise United States Patent (USP) the 5th, 705, No. 628 with multiple nucleic acid purification technology; The 5th, 898, No. 071; The 6th, 534, No. 262; Technology described in No. the 2002/0106686th, U. S. application case and the WO 99758664, the content of these documents is to be incorporated herein by reference) use together.

In one embodiment, the present invention relates to a kind of isolating method of nucleic acid (for example plasmid DNA) that molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the genomic nucleic acids molecular weight, it comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism and iii) reagent, wherein said reagent makes the nucleic acid of cell or organism dissolving and cell be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under cell or organism generation dissolving and cell or organic nucleic acid and the reversible bonded condition of solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic nucleic acid.Solid phase carrier is separated with combination and contact with elution buffer, described elution buffer makes molecular weight be lower than the nucleic acid of genomic nucleic acids molecular weight from the solid phase carrier wash-out, and do not cause genomic nucleic acids, thereby molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the separate nucleic acid of genomic nucleic acids molecular weight from the solid phase carrier wash-out.

In another embodiment, the present invention relates to a kind of method that molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the separate nucleic acid of genomic nucleic acids molecular weight, it comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism lysate and iii) reagent, wherein said reagent makes the nucleic acid of cell or organism lysate be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under the nucleic acid and the reversible bonded condition of solid phase carrier of cell or organism lysate, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic nucleic acid.Solid phase carrier is shifted out and contacts with elution buffer from combination, described elution buffer makes to have the nucleic acid of the molecular weight that is lower than genomic nucleic acids from the solid phase carrier wash-out, and can not cause genomic nucleic acids, thereby molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the separate nucleic acid of genomic nucleic acids molecular weight from the solid phase carrier wash-out.

The present invention relates in addition and a kind of molecular weight in cell or organic genomic nucleic acids and cell or the organism is lower than the method for the separate nucleic acid of genomic nucleic acids molecular weight, and the nucleic acid that wherein has a lower molecular weight is applicable to manually or in the high yield automatization sequence measurement.

(for example the present invention also relates to a kind of isolated cell, prokaryotic cell prokaryocyte, such as the eukaryotic cell of mammalian cell) or the method for the endogenous nucleic acid (for example RNA, DNA) of organism (for example pathogenic agent, such as virus, bacterium, mycobacterium, parasite, fungi).As used herein, " cell or organic endogenous nucleic acid " refers to the nucleic acid that is typically found in cell or the organism (when cell or organism come across occurring in nature, the nucleic acid of finding) in cell or organism.

In one embodiment, the present invention relates to the method for a kind of isolated cell or organic genomic nucleic acids, it comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism and iii) reagent, wherein said reagent makes the nucleic acid of cell or organism dissolving and cell be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under cell or organism generation dissolving and cell or organic genomic nucleic acids and the reversible bonded condition of solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic genomic nucleic acids.Subsequently solid phase carrier is separated with combination, thus isolated cell or organic genomic nucleic acids.Solid phase carrier can be contacted with the elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out.

In one embodiment, the present invention relates to the method for a kind of isolated cell or organic genomic nucleic acids, it comprises cell and makes cell or organism dissolved reagent merge, thereby produces first combination; And described first combination is remained under cell or the organism dissolved condition, thereby produce lysate.Merge lysate and binding buffer liquid, described binding buffer liquid comprises solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid; And reagent, it makes cell or organic nucleic acid be deposited on the solid phase carrier, thereby produces second combination.Described second combination is remained under the reversible bonded condition of cell or organic genomic nucleic acids and solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic genomic nucleic acids.Subsequently solid phase carrier is separated with second combination, thus isolated cell or organic genomic nucleic acids.Described method can further comprise makes solid phase carrier contact with the elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out.

In another embodiment, the method for described herein isolated genes group nucleic acid can be used for the genomic nucleic acids of (for example) isolated viral.In this embodiment, described method comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) virus and iii) reagent, wherein said reagent makes the nucleic acid of described virolysis and virus be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under dissolving and virus takes place virus genomic nucleic acids and the reversible bonded condition of solid phase carrier, thereby generation has the solid phase carrier with the genomic nucleic acids of solid phase carrier bonded virus.Subsequently solid phase carrier is separated with combination, thus the genomic nucleic acids of isolated viral.Solid phase carrier can be contacted with the elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out.Perhaps, virus can be at first with make the reagent of virolysis contact and contact with reagent (it makes nucleic acid of virus be deposited on the solid phase carrier) with solid phase carrier (on its surface in conjunction with the functional group of reversible bind nucleic acid) subsequently.

In the method for isolated genes group nucleic acid, described method can further comprise and add to promote that (for example, when genomic nucleic acids was DNA, reagent was rnase for reagent that non-genomic group nucleic acid removes; When genomic nucleic acids was RNA, reagent was deoxyribonuclease; Remove proteinic proteolytic enzyme (for example Proteinase K)).

Described herein method also can be used for from cell or organism isolation of RNA (for example endogenous RNA).In one embodiment, described method comprises and merges i) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid, ii) cell or organism and iii) reagent, wherein said reagent makes the nucleic acid of cell or organism dissolving and cell be deposited on the solid phase carrier, thereby produces combination.Described combination is remained under cell or organism generation dissolving and cell or organic endogenous nucleic acid and the reversible bonded condition of solid phase carrier, thereby produce the solid phase carrier that has with solid phase carrier bonded cell or organic endogenous nucleic acid.In a specific implementations, subsequently with solid phase carrier with remove or the reagent of dna digestion contacts, and remain on that DNA is removed or digestion and RNA are kept perfectly under the condition of (RNA is not removed or digests).When the reagent that removes DNA is in nucleic acid (for example DNA, RNA) from the solution (for example aqueous solution) of solid phase carrier wash-out, subsequently solid phase carrier is contacted with the reagent that makes RNA be deposited on the solid phase carrier.Subsequently solid phase carrier is separated with combination, thus isolated cell or organic RNA.Solid phase carrier can be contacted with the elution buffer that makes RNA from the solid phase carrier wash-out.

In another embodiment of isolation of RNA, method comprises cell and makes cell or organism dissolved reagent merge, thereby produces first combination; And described first combination is remained under cell or the organism dissolved condition, thereby produce lysate.Merge lysate and binding buffer liquid, described binding buffer liquid comprises solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid; And reagent, it makes cell or organic nucleic acid be deposited on the solid phase carrier, thereby produces second combination.In a specific implementations, subsequently with solid phase carrier with remove or the reagent of dna digestion contacts, and remain on that DNA is removed or digestion and RNA are kept perfectly under the condition of (RNA is not removed or digests).When the reagent that removes DNA is in nucleic acid (for example DNA, RNA) from the solution (for example aqueous solution) of solid phase carrier wash-out, subsequently solid phase carrier is contacted with the reagent that makes RNA be deposited on the solid phase carrier.Subsequently solid phase carrier is separated with combination, thus isolated cell or organic RNA.Solid phase carrier can be contacted with the elution buffer that makes RNA from the solid phase carrier wash-out.

Described herein method be used for nucleotide sequencing the isolated plasmid dna template be easy to automatic mode.Use described reagent to provide another kind of herein and be easy to the separate nucleic acid mode of automatization.

As used herein, term " separation " means the material of discussing and is present in and is different from its naturally occurring physical environment and/or from other nucleic acid molecule or cellular constituent (for example cytolemma, organoid) fully, fully or the part isolated or purified.

As used herein, term " nucleic acid " and " nucleic acid molecule " use with term polynucleotide synonym and it (for example is intended to contain DNA, strand, two strands, covalence closed and lax annular form), RNA (for example, strand and two strands), RNA/DNA heterozygote and polyamide nucleic acid (PNA).

" genomic nucleic acids " refers to genome or the karyomit(e) nucleic acid that is present in cell or the organism.Typically, the molecular weight of genome or karyomit(e) nucleic acid is about 500 kilobase (kilobase; Kb) (for example mycoplasma) is to about 5,000 hundred million base (gigabase; Gb).In specific implementations, the molecular weight of genome or karyomit(e) nucleic acid at about 1000kb to about 250Gb (for example onion (onion)); About 10,000kb is to about 5Gb; About 100,000kb is to about 1Gb; With about 500,000kb to 1,000, in the scope of 000kb.Genomic nucleic acids can be DNA or RNA.

" molecular weight is lower than the nucleic acid of genomic nucleic acids molecular weight in cell or the organism " refers to and is present in the nucleic acid except that genome or karyomit(e) nucleic acid in cell or the organism and can be endogenous or exogenous nucleic acid.The nucleic acid that molecular weight is lower than the genomic nucleic acids molecular weight in cell or the organism has about 1kb usually to about 250, the molecular weight of 000kb (for example BAC).In specific implementations, the nucleic acid that molecular weight is lower than the genomic nucleic acids molecular weight in cell or the organism at about 5kb to about 100,000kb; About 100kb is to about 10,000kb; And about 1000kb is to the scope of about 5000kb.As used herein, " endogenous nucleic acid " (host cell nucleic acid) refers to the nucleic acid that is present in cell or the organism when obtaining cell or organism." exogenous nucleic acid " (external nucleic acid; Recombinant nucleic acid) refers to the nucleic acid that when obtaining cell or organism (for example, transfectional cell or organism, transducer cell or organism), is not present in cell or the organism.Exogenous nucleic acid can owing to be introduced in cell or the organism be introduced into cell or organic ancestors in be present in cell or the organism.Exogenous nucleic acid can directly or indirectly be introduced among cell or organism or its ancestors by the known mode of one of ordinary skill in the art (for example transforming or transfection).The nucleic acid that the example of introducing the exogenous nucleic acid in cell or the organism comprises bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), plasmid, coemid (cosmid), P1 carrier and introduces owing to amplification procedure (for example polymerase chain reaction (PCR)).As used herein, term " plasmid " refers to the double-stranded cyclic DNA material, and it is derived from external source source (for example, in introducing host cell) and can be independent of host chromosome DNA and carries out self replication.Therefore, the cloned DNA that duplicates generation by any above-mentioned carrier contained in described term.The example that is used for nucleic acid is introduced the carrier of cell comprises pUC, pOT, pBluescript, pGEM, pTZ, pBR322, pSC101, pACYC, SuperCos and pWE15.Perhaps, exogenous nucleic acid can be introduced in cell or the organism from the phage (for example coemid, P1) of wrapping up nucleic acid.Other example of " molecular weight is lower than the nucleic acid of genomic nucleic acids molecular weight in cell or the organism " includes, but is not limited to episome nucleic acid, mitochondrial nucleic acid, organelle nucleic acid, RNA, siRNA, plastid, minichromosomes, organelle nucleic acid, primer, viral nucleic acid, bacterial nucleic acid and from the nucleic acid of other pathogenic agent.

" solid phase carrier " is insoluble basically entity under the precipitable condition of nucleic acid.Be applicable to suitable solid phase carrier in the inventive method have enough surface-area with allow effectively in conjunction with and further be characterized by have can reversible bind nucleic acid the surface.Suitable solid phase carrier includes, but is not limited to particulate, fiber, bead and upholder, nucleic acid is had avidity for it and it can embody the multiple shape of rule or irregular form, as long as the carrier that the surface-area of described shape maximization solid phase and embodiment are subjected to the microscale operation.In one embodiment, solid phase carrier is a paramagnetism, for example paramagnetic particles (magnetic response).In another embodiment, solid phase carrier comprises that functional group's bag is by the surface.For instance, solid phase carrier can be the amine bag and is sealed the carboxyl bag by paramagnetic particles by paramagnetic particles, carboxyl bag by paramagnetic particles or capsule.

As used herein, " paramagnetic particles " refer to response external magnetic field (the plastic cement pipe or the microtitration board binding that for example have embedding rare earth (for example neodymium) magnet) and after removing magnetic field the particulate of demagnetization.Therefore, can use magnet that paramagnetic particles is effectively separated with solution, but its resuspending is not easily taking place under the magnetic induced accumulative situation.Preferred paramagnetic particles comprises the nuclear that is rich in magnetite by straight polymer shell capsule envelope.Suitable paramagnetic particles comprises about 20%-35% magnetite/capsule envelope ratio.For instance, the magnetic particle that comprises about 23%, 25%, 28%, 30%, 32% or 34% magnetite/capsule envelope ratio is applicable among the present invention.Comprise the weak attraction that magnetic particle less than about 20% ratio only is subjected to being used to realizing the magnet of magnetic resolution.Decide on the character of host cell, the viscosity of cell growth and the character (for example high or low copy) of carrier, should consider to comprise the paramagnetic particles of higher percent magnetite.Use and do not have the iron of exposure or Fe in its surface ₃O ₄Capsule envelope paramagnetic particles eliminate the possibility of disturbing the polysaccharase function at iron in some subsequent operations of DNA isolation.Yet magnetite nuclear is big more, and the possibility that the capsule envelope is leaked (for example discharging ferric oxide) is high more.Be applicable to that the suitable paramagnetic particles among the present invention can be available from (for example) Bangs Laboratories Inc., Fishers, IN are (for example, Carboxylicesters modification capsule envelope magnetic microsphere), Agencourt Biosciences and Seradyn.

Suitable paramagnetic particles should have specific size so that its (for example) separates not difficult from solution by magnetic means or filtration.In addition, preferred paramagnetic particles should be excessive so that its surface-area is through minimizing or it is unsuitable for the microscale operation.Be fit to size in about 0.1 mean diameter to the scope of about 100 mean diameters.Preferred size is about 1.0 mean diameters.Suitable magnetic particle is available from PerSeptive Diagnostics and be known as BioMag COOH.

As used herein, term " functional group's bag is by the surface " refers to the surface through the part bag quilt of reversible bind nucleic acid (for example DNA, RNA or polyamide nucleic acid (PNA)).An example is the surface of the part bag quilt through having free functional group separately, and described free functional group combines with the amino or the particulate of aminosilane; Therefore, microparticle surfaces is through containing the part bag quilt of functional group.The biological affinity adsorbent of preliminary precipitation nucleic acid (for example polyalkylene glycol preliminary precipitation DNA) serves as in functional group.In one embodiment, functional group is a carboxylic acid.Suitable part with free carboxy acid functional group is the Succinic Acid part, and amine bond and the second carboxylic acid not bond of one of them hydroxy-acid group by amido linkage and aminosilane makes free carboxylic acid groups connect or be tethered on the surface of paramagnetic particles.The carboxylic acid bag by magnetic particle available from PerSeptive Diagnostics (BioMag COOH).Functional group bag with reversible bind nucleic acid molecule is had functional group's bag by the magnetic response solid phase carrier on surface by the suitable solid phase carrier on surface for (for example), such as (but being not limited to) amino bag by, carboxyl bag quilt and capsule envelope carboxyl bag by paramagnetic particles.

The lysate that suitable initial substance includes body, cell (complete or full cell), tissue (for example solid tissue, paraffin-embedded tissue), cell lysates (cell in growth or substratum) and prepared by these cells or organism.In one embodiment, initial substance is for containing the fresh or frozen blood (for example whole blood) and the serum of Citrate trianion, EDTA or Vitrum AB coagulant.In another embodiment, initial substance is the cheek cell.Suitably initial substance comprises available from the cell of Mammals (being people, primate (chimpanzee), equine species, Canis animals, feline, bovid, porcine animals, murine) tissue or body fluid with by the lysate of these cell preparation.Therefore, can use and have nucleic acid (for example, genomic nucleic acids; Molecular weight is lower than the nucleic acid of genomic nucleic acids molecular weight) the cell or the organism of any kind.The example that is applicable to the cell in the inventive method includes, but is not limited to mammalian cell (hemocyte for example, such as complete blood cell), bacterial cell (intestinal bacteria (E.Coli) for example, such as DH5, DH10B, DH12S, C600 or XL-1 Blue), yeast cell, vegetable cell, histocyte is (for example from nematode (C.elegans), mouse tail, human bioptic cell) and contain exogenous nucleic acid (recombinant DNA for example, DNA of bacteria or replicative DNA) host cell, its through target with host cell chromosome DNA and other host cell biomolecule molecular separation.Organic example comprises virus (for example hepatitis virus, human immunodeficiency virus, simplexvirus, influenza virus), bacterium, mycobacterium (mycobacterium bovis (M.bovis BCG), leprosy bacillus (M.leprae), mycobacterium tuberculosis (M.tuberculosis)), fungi (for example yeast, such as yeast saccharomyces cerevisiae (C.cerevisiae), Candida albicans (C.albicans)) and parasite (for example leishmania (Leishmania)).Perhaps, the initial substance lysate of these cell preparation of can serving as reasons.

As used herein, " host cell " or " host's organism " is exogenous nucleic acid can be introduced wherein, thereby produces host cell or the organic any cell that contains exogenous nucleic acid except that host cell or organism nucleic acid.As used herein, term " host cell or organism nucleic acid " and " endogenous nucleic acid " refer to the nucleic acid substances (for example genome or karyomit(e) nucleic acid) that is present in host cell or the organism when obtaining cell or organism.As used herein, term " external source " refers to the nucleic acid (for example plasmid) except that host cell or organism nucleic acid; Exogenous nucleic acid can owing to be introduced in host cell or the organism be introduced into host cell or organic ancestors in be present in host cell or the organism.Therefore, for example, particular host cell or organic exogenous nucleic acid material are non-endogenous nucleic acid material (be not present in wherein or be not present among its ancestors when time in acquisition host cell or the organism).Suitably host cell includes, but is not limited to bacterial cell, yeast cell, vegetable cell and mammalian cell.

As described herein, the invention has the advantages that and to use the minimized number step cell or organic genomic nucleic acids and molecular weight to be lower than the cell or the organic separate nucleic acid of genomic nucleic acids molecular weight.As described herein, directly pair cell or organism culture separate, and need not granulation cell or organism.In addition, but described method pair cell or organism lysate implement, wherein the inventive method makes it needn't clarify lysate cell or organic genomic nucleic acids and molecular weight are lower than the separate nucleic acid of endogenous nucleic acid molecular weight.Basically all nucleic acid are in the embodiment of endogenous nucleic acid (for example DNA, RNA) in cell or organism, and described herein method can be used for the endogenous nucleic acid of particular types (genomic nucleic acids for example; Total RNA) separates with cell or organism.

As used herein, " lysate " is for containing the solution of cell or organism composition, it contains genomic nucleic acids and has than the more low-molecular-weight nucleic acid of genomic nucleic acids, and its cytolemma is destroyed by any way, makes cell or organic inclusion (comprising nucleic acid wherein) all be in the solution." clarification lysate " for cell or organic karyomit(e) or genomic nucleic acids, protein and film (such as) lysate that removes by chemical treatment or centrifugal lysate.Use currently known methods dissolved cell or organism, thereby preparation is suitable for the mixture that the inventive method is used.For instance, can use chemical mode (for example, alkalescence or alkalescence and anionic washing agent handle), etc. open and impact or physical damage (for example homogenizing) dissolved cell or organism.

As used herein, term " dissolving host cell suspension " or " dissolving host organism suspension " refer to and comprise host cell or organic suspension, its film by any way (for example, chemistry (handling) such as alkalescence or alkalescence and anionic washing agent, etc. open impact or carry out physical damage by homogenizing) destroy; This suspension is host cell biomolecule molecule, cellular constituent and through destroying the mixture of film fragment.In one embodiment, be applicable to that dissolving host cell or organism suspension among the present invention are by host cell or organism are prepared with alkalescence and anionic washing agent (for example sodium lauryl sulphate (SDS)) solution (for example, 0.2 N NaOH, 1%SDS) contact.According to circumstances, N,O-Diacetylmuramidase can be included in the dissolving damping fluid.The existence of anionic washing agent is used for producing the anti-protein environment by the proteinic effective charge that neutralizes in the solvent soln, thereby minimize it functional group is wrapped by the attraction on paramagnetic particles surface.In one embodiment, dissolving host cell or organism suspension are without neutralization.According to circumstances, rnase can be added in host cell or the organism lysate decomposing host cell RNA, thereby DNA is combined with the magnetic particle that does not contain or be substantially free of RNA.

The method according to this invention merges cell or organism and solid phase carrier and reagent, and wherein said reagent makes cell or organic nucleic acid is non-specific and reversibly combine with solid phase carrier.

" non-specific nucleic acid combination " refers to the combination that magnetic particle is had the different IPs acid molecule of about identical affinity, but the size of nucleotide sequence or different IPs acid molecule there are differences.As used herein, " promote absorption " refers to and uses precipitation reagent (for example polyalkylene glycol) to promote dna molecular material (it is initially in the mixture) precipitation and be adsorbed in process on the surface of solid phase carriers subsequently.Two kinds of the reversible interaction of gained and (for example) are different in conjunction with the interaction between the collocation thing (for example streptavidin/vitamin H, antibody/antigen or sequence-specific interact), and it is generally used for separating according to its composition or sequence the purpose of specific biological molecules.

" nucleic acid precipitation reagent " or " nucleic acid precipitation agent " or " aggregating agent prepared therefrom (crowding reagent) " is for to make cell or organic nucleic acid leave the composition of solution.Suitable precipitation agent comprises alcohol (for example short chain alcohol, such as ethanol or Virahol) and polyol (for example polyalkylene glycol).The nucleic acid precipitation reagent can comprise one or more these reagent.The nucleic acid precipitation reagent exists with non-specific and reversibly the nucleic acid of cell is incorporated on the solid phase carrier with enough concentration.

Be applicable to that suitable alcohol (for example ethanol, the Virahol) concentration (ultimate density) in the inventive method is about 40% to about 60%; About 45% to about 55%; With about 50% to about 54%.

Suitably polyalkylene glycol comprises polyoxyethylene glycol (PEG) and polypropylene glycol.Suitable PEG can be available from Sigma (Sigma) (Sigma Chemical Co., St Louis MO.; Molecular weight 8000 does not contain deoxyribonuclease and rnase (Dnase and Rnase fee), catalog number (Cat.No.) 25322-68-3).The molecular weight of polyoxyethylene glycol (PEG) can be about 6000 to about 10,000, about 6000 to about 8000, about 7000 to about 9000, about 8000 to about 10,000 scope.In a specific implementations, use molecular weight to be about 8000 PEG.In general, the existence of PEG provides hydroholic solution, and it forces the hydrophilic core acid molecule to leave solution.In one embodiment, PEG concentration is about 5% to about 20%.In other embodiments, PEG concentration is about 7% to about 18%; About 9% to about 16%; With about 10% to about 15% scope.

As indicated above, be in cell or the organic embodiment at initial substance, reagent through allotment so that the dissolving of cell or organism.Can use multiple dissolved constituent destroy film (such as alkalescence, alkalescence and anionic washing agent handle or etc. open impact).Make that cell or organism dissolved reagent can be including (for example) sodium hydroxide (NaOH), sodium lauryl sulphate (SDS), Triton, sodium lauryl sarcosinate and/or guanidinium isothiocyanates.In one embodiment, the dissolved constituent of reagent is alkalescence (NaOH) solution and/or anionic washing agent (for example sodium lauryl sulphate (SDS)) solution (for example, in the time of in adding cell to, ultimate density is 0.2 N NaOH, 1%SDS).According to circumstances, N,O-Diacetylmuramidase can be included in the dissolved constituent of first reagent.The existence of anionic washing agent is used for producing the anti-protein environment by the proteinic effective charge that neutralizes in the dissolved constituent, thereby minimizes its attraction to solid phase carrier (functional group wraps by paramagnetic particles) surface.According to circumstances, can be with rnase (for example, 1.75ng/ μ l rnase/ddH ₂O) add in the dissolved constituent decomposing host cell RNA, thereby DNA is combined with the solid phase carrier that does not contain or be substantially free of RNA.The necessity that comprises the rnase step will be largely by the size decision of nucleic acid substances, and described nucleic acid substances separates in target precipitates with the specific nucleic acid that is being carried out.For instance, if selected separation condition is suitable for separating the nucleic acid that comprises at least 4,000 base pair, RNA unlikely becomes obvious pollutent so.

Used reagent is suitable for nucleic acid is separated with cell or organism in the aforesaid method.This reagent contains nucleic acid precipitation agent and solid phase carrier, and also can be through allotment so that one or more cell or organism dissolving.The nucleic acid precipitation agent has enough concentration so that cell or organic nucleic acid precipitation.Solid phase carrier in this reagent contains the surface in conjunction with cell or organic nucleic acid.Can comprise the reagent component that is single agents or independent component form.Described component can simultaneously or merge with cell successively.The order that merges combined composition is not crucial.Comprise in the reagent described in the property quality and quantity such as above-mentioned method of component.Reagent is adjustable to be conc forms, and making needs dilution to obtain function and/or the concentration described in this paper method.

According to circumstances, salt can be added in the reagent so that the nucleic acid of cell is deposited on the solid phase carrier.Be suitable for promoting that the suitable salt through the target isolated nucleic acid molecule is adsorbed on the magnetic response particulate comprises sodium-chlor (NaCl), lithium chloride (LiCl), bariumchloride (BaCl ₂), potassium (KCl), calcium chloride (CaCl ₂), magnesium chloride (MgCl ₂) and cesium chloride (CsCl).In one embodiment, use sodium-chlor.In general, the existence of salt is used to minimize the negative charge repulsion of nucleic acid molecule.The wide region indication that is applicable to the salt in the described method also can use many other salt and suitable content to be determined by rule of thumb by one of ordinary skill in the art.Salt concn can be about 0.1M to about 0.5M; About 0.15M is to about 0.4M; With about 2M to about 4M.

Under high salt concentration (for example, with the high ionic strength synonym), suitable paramagnetic particles will be adsorbed the dna fragmentation of all sizes.Term " high salt concentration " refers to the salt concn greater than about 0.5M.Under " low salt concn " (or low ionic strength), its hint concentration is less than about 0.2M as used herein, even the DNA of virtually any size can not precipitate (Lis yet in the presence of the PEG concentration up to 12% (weight/volume) basically, John T, Methods in Enzymology 65:437-353 (1980)).Other component can be added in the reagent.In one embodiment, rnase is added in the nucleic acid precipitation agent.

The method according to this invention is by being shifted out the separation that realizes cell or organic nucleic acid molecule with the nucleic acid bag by solid phase carrier from combination.Can (for example) by vacuum filtration, centrifugal or to attract solid phase carrier (for example paramagnetic particles) solid phase carrier (for example paramagnetic particles) is reclaimed from first combination by applying magnetic field.The preferred magnetic means (such as the magnetic field that applies at least 1000 Gausses (Gauss)) of using is with paramagnetic particles and solution separating.Yet, can use known other method of those skilled in the art (for example vacuum filtration or centrifugal) that magnetic particle is shifted out from supernatant liquor.Removable subsequently surplus solution stays and has the solid phase carrier that is adsorbed in its lip-deep cell or organic nucleic acid.In case, can reclaim the nucleic acid that the molecular weight that is adsorbed on the solid phase carrier is lower than the molecular weight of cell or organic genomic nucleic acids by solid phase carrier is contacted with suitable elution buffer from mixture separation.Therefore, produce 1) in elution buffer, comprise the solution and 2 that molecular weight is lower than the nucleic acid molecule of genomic nucleic acids molecular weight) in conjunction with the solid phase carrier of cell or organic genomic nucleic acids.

In one embodiment, be applicable to that being fit in the inventive method " elution buffer " is lower than the cell of molecular weight of cell or organic genomic nucleic acids or the damping fluid of organic nucleic acid for the selective elution molecular weight.Basically all nucleic acid are in the embodiment of genomic nucleic acids (for example DNA, RNA) in cell or organism, and suitable elution buffer is the damping fluid from solid phase carrier wash-out genomic nucleic acids.In one embodiment, be applicable to that the suitable elution buffer among the present invention can be water or aforesaid any aqueous solution, wherein nucleic acid precipitation agent (for example Virahol and/or PEG) concentration is lower than cell or organic nucleic acid that molecular weight is lower than cell or organic genomic nucleic acids molecular weight and combines required concentration with solid phase carrier.For instance, useful buffer reagent includes, but is not limited to TRIS-HCl, Tutofusin tris acetate (Tris acetate), sucrose (20%) and methane amide (100%) solution.When use being fit to the low ionic strength elution buffer, can be rapidly (for example, in 30 seconds or shorter time) generation molecular weight be lower than the cell of cell or organic genomic nucleic acids molecular weight or organic nucleic acid wash-out from solid phase carrier.In case bonded nucleic acid is by wash-out, just with cell or organic genomic nucleic acids institute bonded solid phase carrier with comprise the elution buffer that molecular weight is lower than the nucleic acid of genomic nucleic acids molecular weight and separate.Solid phase carrier can be contacted with the suitable elution buffer that makes genomic nucleic acids from the solid phase carrier wash-out subsequently.

According to circumstances, can (for example contact with the reagent of removal and/or dissolved impurity by making to have with the paramagnetic particles of its bonded nucleic acid, by particulate is contacted with suitable washing buffer solution) (for example remove impurity, host cell component, specific nucleic acid, protein, metabolite, chemical agent or cell debris), then particulate institute bonded nucleic acid is separated with solid phase carrier.The reagent of removal and/or dissolved impurity can be the reagent of dissolving specific nucleic acid, such as DNA (for example deoxyribonuclease), RNA (for example rnase) or protein (for example Proteinase K).Perhaps, the reagent of dissolved impurity can be " lavation buffer solution " or is included in the lavation buffer solution, its for dissolving removal directly combines with particulate or with the associating impurity of adsorbed nucleic acid, but insoluble desorb invests the composition of the nucleic acid on the solid phase.The pH value of lavation buffer solution and solute are formed and concentration can change according to the dopant type that expection exists.For instance, ethanol (for example 70%) illustration is applicable to the preferred lavation buffer solution of removing excessive PEG and salt.Also available more than one washing buffer solution wash the magnetic particle with institute's bind nucleic acid.On demand repeatedly (for example three to five times) washing particulate is to remove required impurity.Yet, preferably limit the loss of washing times bind nucleic acid output to minimize.Be fit to washing buffer solution and have some specific characters.At first, washing buffer solution should have enough high salt concentrations (enough high ionic strengths) so that can not wash out from particulate with magnetic particle bonded nucleic acid, and still combines with particulate.Suitable salt concn is greater than about 0.1M and be preferably about 0.5M.Secondly, buffered soln through selecting so that with nucleic acid or the dissolving of particulate bonded impurity.The pH value of buffered soln and solute are formed and concentration can change according to the dopant type that expection can exist.Suitable washing soln comprises following: 0.5 * 5 SSC; 100mM ammonium sulfate, 400mM Tris pH 9,25mM MgCl ₂With 1% bovine serum albumin (BSA); With 0.5M NaCl.In one embodiment, wash buffered soln and comprise 25 mM Tutofusin tris acetates (pH 7.8), 100mM potassium acetate (KOAc), 10mM magnesium acetate (Mg ₂OAc) and 1mM dithiothreitol (DTT) (DTT).In another embodiment, washing soln comprises 2%SDS, 10% tween (Tween) and/or 10%Triton.

The starting soln of high-quality nucleic acid preparation and multicomponent and complicacy be separated into basic technology in the molecular biology.Therefore, because described research herein, nowadays exist the isolated molecule amount to be lower than nucleic acid molecule novel of genomic nucleic acids molecular weight and be easy to automated method.In one embodiment, by various product transfer device reagent is added in cell or the organism.In another embodiment, first reagent is added in a plurality of samples (for example at least 6,12,24,96,384 or 1536 samples) simultaneously, each sample contains one or more cell or organism.In another embodiment, first reagent is delivered in a plurality of samples (for example at least 6,12,24,96,384 or 1536 samples) successively, each sample contains one or more cell or organism.The present invention includes the method for analyzing a plurality of nucleic acid samples.Described method comprises to be provided by isolating a plurality of nucleic acid samples of described method herein and analyzes described sample (for example, sample being carried out sequential analysis).

In another embodiment, make the isolating nucleic acid of one or more sample carry out other analysis (for example, sequential analysis).Needing can be used for the molecular biology of high-quality nucleic acid to use by institute's isolating nucleic acid of revealing method, the for example preparation of dna sequencing template, microinjection, the transfection of mammalian cell or conversion, the external of rna probe synthesized, the reverse transcription clone, the cDNA library construction, pcr amplification or gene therapy study, and having more not other application of severity specification of quality, it includes, but is not limited to transform, restriction endonuclease or microarray analysis, selectivity RNA precipitation, external transposition, the separation of polynary pcr amplification product, external siRNA, the RNAi hair clip, the preparation of dna probe and primer and stripper plate (detemplate) scheme.

The present invention is also contained and is comprised the test kit that one or more is applicable to the reagent in the described method herein.In one embodiment, described test kit comprises to make cell or organism dissolved reagent (for example dissolving damping fluid, such as TRIS-HCl, washing agent, sodium lauryl sulphate, Triton X-100, EDTA) and makes nucleic acid and solid phase carrier bonded reagent (for example binding buffer liquid).In a specific implementations, binding buffer liquid comprises the nucleic acid precipitation reagent, such as PEG; Salt is such as NaI; And/or solid phase carrier, such as magnetic particle.Test kit can further comprise the reagent of dissolved impurity.In one embodiment, the reagent of dissolved impurity is the reagent (for example Proteinase K) of digesting protein.Test kit can further comprise other damping fluid, such as (one or more) lavation buffer solution (for example PEG, urea, NaCl, Tris-HCl) and/or (one or more) elution buffer.In a specific implementations, test kit comprises the reagent (for example Proteinase K) and the lavation buffer solution (for example PEG, urea) of dissolving damping fluid (for example TRIS, washing agent), binding buffer liquid (for example nucleic acid precipitation reagent (such as PEG) and/or salt (such as NaI) and magnetic particle), digesting protein.But one or more reagent can be through merging in the reagent Individual existence of test kit or the test kit.Other component that can be provided in the test kit comprises various product container (for example microtiter plate), the various product fixer of magnetic r (for example microtitration board binding) and various product transfer device (for example hyperchannel transfer pipet).Test kit can further comprise about using the explanation of test kit and its component.

Embodiment

Embodiment 1 is used for isolated cell and has a embodiment than the scheme of the more low-molecular-weight nucleic acid of genomic nucleic acids

Following scheme is illustrated among Fig. 1.

The growth of bacterial cultures

1. 200L being contained suitable antibiotic 2 * YT bacterial growth media is drawn in each hole of culture plate (catalog number (Cat.No.) 3750) at the bottom of 300lCostar 96 hole circles with dropper.

2. each hole is implanted the single plasmid that contains the intestinal bacteria bacterial colony (DH10B, DH5alpha:Invitrogen).Grown culture can directly be implanted from agar lawn or glycerine storing solution.

3. with perforated lid covering culture plate and 37 ℃ of following strong vibrations (for example 300rpm) 16 hours.

Purifying procedure

1. 60L paramagnetism dissolving damping fluid (0.4 N NaOH, 2%SDS, 0.0016% solid Agencourt COOH magnetic particle) solution is added in each hole of source culture plate and vibration or tilt to mix.

2. 60L A washing soln (100% Virahol) is added in the sample.Carrying out 15 times after adding the A washing soln tilts to mix.The visual tup of inclination mixing condition hole (tup orifice) and changing.

3. the source culture plate is placed on magnetic SPRIplate96-R (TM) (catalog number (Cat.No.) AgencourtBiosciences) last 5 minute with from the solution separating bead.

4. from the sucking-off of purpose culture plate and discard settled solution, place it in simultaneously on the SPRIplate96-R (TM) (toroidal magnet).

5. 200L B washing soln (70% ethanol) is allocated in each hole of purpose culture plate as washing soln.

6. remove washing with alcohol solution and step is repeated four times.

The purpose culture plate is following dry 30 minutes at 37 ℃ 7..

8. with 40L elution buffer (ddH ₂The O+1.75ng/l ribonuclease A) add in each hole of culture plate and vibration.Suggestion SILVER REAGENT water is as elution buffer, and vortex or shaking culture plate are so that the wash-out generation after adding elution buffer or waiting for 10 minutes.

The order-checking program:

React interpolation for 1/24th * BigDye:

The 60l purify DNA

The 1lBigDye mixed solution

The BigDye mixed solution

5 parts of BigDye terminators

1 part of 200m-M13-21 primer

4 portions of 15 * BigDye damping fluids

Cycle sequencing

95 ℃, 2 minutes

50 round-robin (54 ℃, 15 seconds; 60 ℃, 2.5 minutes; 95 ℃, 5 seconds)

4 ℃, up to purifying

Use arbitrary standard EtOH precipitin reaction of Agencourt CleanSeq test kit (catalog number (Cat.No.) 000121 and 000222) to come purifying sequencing reaction thing.

In 30l deionized water (ddH ₂O) in wash-out CleanSeq product precipitation and be placed on the detection of checking order on ABI 3700 or 3730.

Use the POP5 polymkeric substance to produce 90% percent of pass (should in bp 100-300, be averaged) and 625 Phred20 bases carrying out 11,000 above sequence readings on the ABI 3700 to phred 20 by reading.

Has separation in embodiment 2 bacterial cells than the more low-molecular-weight exogenous nucleic acid of genomic nucleic acids

Method and material

Clone and purifying: the chimpanzee genomic dna is sheared,, and cloned in the pOT bacteria carrier with T4 polysaccharase and the terminal reparation of Klenow (NEB).With DH10B cell (Invitrogen) electroporation and be applied on 25 μ g/ml paraxin (chloramphenicol) agar and grow overnight.Pick up bacterium colony in 200 μ l 2XYT, the 50 μ g/ml paraxin meat soups and grew 16 hours with Gentix Qpix.To clone thing on Beckman FX robot platform in the grown cultures plate purifying.

Purification process

Will be herein the method (McPrep) described in No. the 6th, 534,262, described method (OneStep Prep) and the United States Patent (USP) compare.

Use two kinds of different purification process (McPrep and described single stage purification process) to come 24 samples of purifying herein.Controlled sample is loaded on the sepharose relatively to reclaim (Fig. 2).

Except that 60 μ l, 100% Virahol (or 100 μ l 40%PEG), add to 60 μ l paramagnetisms dissolving damping fluids (0.4 N NaOH, 2%SDS, 160 mg/litre Seradyn COOH paramagnetism beads) in the cell culture and the mixing 15 times of tilting.Sample is gone up separation at Agencourt magnet culture plate (1000 Gauss) reach 6 minutes.Remove supernatant liquor and with 70% ethanol with bead isolation flushing 5 times.Using 1.75ng/ μ l ribonuclease A/ddH ₂Behind O (Sigma) wash-out, go up the operation sample at 96 E-Gel (Invitrogen) and reclaim (Fig. 2) to assess relative DNA.Also sample is carried out that Pico Green analyzes (Molecular Probes) and average dna is recovered as 20ng/ μ l.The record sample average conductivity with by compile from whole 40 μ l eluents in 10 holes assess from the growth meat soup any salt pollution.Electric conductivity is less than 20 μ s/cm.

Sequencing reaction is set:

With diluted sample in 40 μ l 1.75ng/ μ l rnase/ddH ₂Carry out robot among the O and under 2Hz and stir 20 round-robin .5cm.Sucking-off 3 μ l DNA and place it in 0.5 μ l (1/3X BigDye) reagent and add in 3 picomole-21 primer.Cover reactant and in 384 well culture plates, carry out cycle sequencing with mineral oil.According to manufacturers's suggestion sample is carried out cycle sequencing.The sequencing reaction thing carried out purifying with the CleanSeq test kit of Agencourt and at 15 μ l ddH ₂Wash-out and carry out heat seal among the O is loaded on the ABI 3700 then.

Detect:

Use ABI3700 order-checking platform and 1/48thX BigDye V3.1 order-checking chemistry and POP5 polymkeric substance to produce the dna sequencing data.Be injected at 30 seconds and carry out electrophoresis under 5800 V.

The result:

Produce 12672 order-checking readings (table 1) by 6336 purification of samples.For 48 times of dilution BigDye order-checking chemistry, the high percent of pass that is obtained, high signal intensity and high mean P hred 20 base pairs people such as (, Genome Research, 8:175-185 (1998)) Ewing are unusual.The column diagram of reading performance is drawn among Fig. 3.

Fig. 2 shows 96 hole sepharoses with 13 row * 8 row.The 13rd classifies 200ng pGEM 3.2kb carrier (Promega) as.Positive electrode is in the picture bottom.To prepare sample wash-out and 10 μ l are loaded on the gel in the various elution buffers of 40 μ l.As seen the 3-5kb plasmid on the gel does not have bacillus coli gene group DNA and stays sign in the agarose hole.Visible RNA in without the hole (row F) of 1.75ng/ μ l rnase wash-out.

With respect to using the McPrep method, as seen use single step process to be recovered to more plasmid DNA (row A is to row B and go C to row D).This is contemplated to the McPrep method needs bacillus coli gene group DNA to be incorporated on one group of bead and the supernatant liquor that is rich in plasmid is moved in another culture plate to combine with second mixture.This supernatant liquor removes step makes it be difficult to sucking-off 100% plasmid supernatant liquor under the situation that does not upset the genomic dna magnetic beads, and therefore there is the DNA loss in expection.

Row A:10 μ l/40 μ l McPrep

Row B:10 μ l/40 μ l OneStep Prep

Row C:10 μ l/40 μ l McPrep

Row D:10 μ l/40 μ l OneStep Prep

Row E and F: the 10 μ l/40 μ l OneStep that have and do not have rnase

Row G: the 10 μ l/40 μ l McPrep that do not have rnase

Use 3 μ l to carry out the BigDye order-checking through the wash-out plasmid DNA.Phred measures electric long distance image (Eletropherogram) reading quality people such as (, Genome Research, 8:175-185 (1998)) Ewing by operation.Phred 30 bases have 1/1000 error rate, and Phred 40 bases have 1/10,000 error rate.The phred 20 base counting numbers that each reading is produced are standard sequencing quality tolerance (table).

Percent of pass is through being defined as the number of readings per taken/total indicator reding that satisfies by standard

The mean number of the Phred 30 of per 384 well culture plates of P30=

The mean number of the Phred 20 of per 384 well culture plates of P20=

The mean number of the continuous P hred 20 of per 384 well culture plates of CP20=

The mean number of the Phred 15 of per 384 well culture plates of P15=

The mean P hred mark of the whole readings of Qual=: the mean value of 384 well culture plates

Phred20 quality from the 200bp window of bp 100 to bp 300 should be averaged by standard-reading.

SigA=is for 96 readings, the average relative fluorescence unit in the A channel

SigG=is for 96 readings, the average relative fluorescence unit in the G passage

SigC=is for 96 readings, the average relative fluorescence unit in the C-channel

SigT=is for 96 readings, the average relative fluorescence unit in the T passage

Sequence B arcode	By	Altogether	Pass through %	P30	P20	CP20	P15	Qual	Length	SigA	SigG	SigC	SigT
														000048032741(KF)	351	384	91.41	576	669	540	706	47	763	188	134	177	187
000028713241(KN)	360	384	93.75	528	626	482	670	46	743	149	118	161	158
														000028713341(KP)	345	384	89.84	534	625	489	663	46	736	51	33	46	50
000028713641(HK)	345	384	89.84	538	620	509	660	44	744	93	64	85	103
														000028714941(GQ)	353	384	91.93	552	639	515	677	43	741	202	151	191	208
000028715041(KF)	336	384	87.50	555	654	520	694	46	754	197	125	168	186
														000048032841(KF)	350	384	91.15	568	659	527	696	46	754	165	114	146	162
000028712941(KN)	357	384	92.97	588	681	560	111	47	778	188	156	173	197
														000028713041(KH)	369	384	96.09	585	670	545	705	47	768	94	56	63	82
000028713141(KJ)	341	384	88.80	538	627	521	663	47	726	113	79	110	148
														000028713441(KP)	341	384	88.80	569	665	534	703	46	760	115	75	98	114
000028713541(KN)	339	384	88.28	528	632	.487	677	46	755	268	217	295	292
														000028713741(GZ)	347	384	90.36	549	639	515	677	44	741	165	117	143	146
000028713941(HK)	349	384	90.89	548	638	522	676	45	740	177	121	148	177
														000028714141(KN)	352	384	91.67	498	598	447	643	45	727	108	83	113	114
000028714241(KK)	347	384	90.36	548	646	516	685	46	747	120	83	99	127
														000028714441(GY)	350	384	91.15	529	621	503	660	46	727	138	98	124	135
000028714541(GQ)	343	384	89.32	525	620	483	663	43	738	194	118	182	184
														000028714G41(KF)	311	384	80.99	540	638	481	678	45	747	146	89	119	141
000028714741(XB)	351	384	91.41	488	612	371	661	38	726	370	120	128	178
														000028714841(KF)	333	384	86.72	569	666	527	706	47	759	205	122	161	191
000028715141(HH)	363	384	94.53	536	618	507	652	4.6	712	191	129	157	201
														000028715541(GQ)	358	384	93.23	532	619	505	660	43	731	110	86	102	110
000028715641(KR)	362	384	94.27	530	612	495	645	46	710	51	45	65	65
														000028712841(HG)	361	384	94.01	537	634	504	675	44	741	360	220	335	339
000028714041(KF)	349	384	90.89	542	637	499	677	45	746	77	49	61	75
														000028715241(GW)	359	384	93.49	497	580	462	618	43	699	132	67	102	117
000028715441(HH)	345	384	89.84	487	570	450	610	43	696	104	70	81	109
														000028715741(KS)	354	384	92.19	464	546	393	585	45	675	39	29	39	38
000028713841(HA)	358	384	93.23	534	623	507	660	46	721	126	96	144	167
														000628712741(GR)	326	384	84.90	434	520	401	561	41	674	76	53	64	73
000028714341(GR)	328	384	85.42	481	570	446	613	42	704	99	70	84	96
														000028715341(GR)	334	384	86.98	478	563	455	603	42	698	114	69	84	99
	11467	12672	90.49	531	623	492	662	45	733	143	99	129	145

Fig. 3 is by the Phred 20 (redness) of reading generation and the column diagram of Phred 30 (black) base.Y-axis is a number of readings per taken, and X-axis is that increment is phred 20 boxes of 50bp.

Has separation in the embodiment 3 horse complete blood cell than the more low-molecular-weight nucleic acid of genomic nucleic acids

Material and method

The source

Acquisition is the horse whole blood of 1: 1 ratio with Alsevers antithrombotics (2.05% dextrose, 0.5% Trisodium Citrate, 0.055% citric acid, 0.42% sodium-chlor).

Purification process

Except that 80 μ l, 100% Virahol, add to 60 μ l paramagnetisms dissolving damping fluids (0.4 NaOH, 2%SDS, 160 mg/litre Seradyn COOH paramagnetism beads) in the cell culture and the mixing 15 times of tilting.Sample is gone up separation at Agencourt magnet culture plate (1000 Gauss) reach 15 minutes.Remove supernatant liquor and with 70% ethanol with bead isolation flushing 5 times.Using ddH ₂Behind O (Sigma) wash-out, sample is gone up operation in 96 E-Gel (Invitrogen) reclaim (Fig. 4) to assess relative DNA.Also sample is carried out that Pico Green analyzes (Molecular Probes) and average dna is recovered as 0.5ng/ μ l (Fig. 5).Confirm DNA quality (Fig. 6) with its suitability for polymerase chain reaction (PCR) (common subsequent applications).

Fig. 4 shows two parts of gDNA by the preparation of 50 μ l horse blood.Fig. 5 shows that 8 PicoGreen by the sample of gDNA preparation analyze.Fig. 6 shows the grads PCR (using the Y3B19 mark of expection amplicon size as 225bp) of above-mentioned prepared gDNA.

Embodiment 4 use single step dissolvings and combined techniques with genomic dna from the horse separation of whole blood

1. 50 μ l horse blood are placed in the porous culture plate.

2. add 200 μ l dissolving damping fluid (20%PEG1000,1 M NaCl, 0.5%SDS, 1%TritonX-100,10mM EDTA, 20mM Tris HCl, 0.29% solid magnetic bead), mix by drawing 10 times.Cultivated 5 minutes.

3. be placed on the magnetic sheet about 5 minutes.

4. remove supernatant liquor, stay bead.

With the bead resuspending in 250 μ l lavation buffer solutions (6M urea/14%PEG1000,1 M NaCl).Wait for 5 minutes, do not have magnetic bases.

6. be placed on magnetic bases last 5 minute.Remove supernatant liquor.

7. repeating step 5 and 6.

With the bead resuspending in 70% ethanol and with bead magnetic resolution 3 times.

9. air-dry 5-10 minute.

10. add the water that 50 μ l-200 μ l do not contain nuclease, 60 ℃ of-70 ℃ of following preheatings.Mixed 10 minutes.

11. culture plate is placed on the magnetic sheet with bead isolation.Shift out through eluted dna.

The result

DNA is loaded on the 0.8% agarose e-gel.Use OD 260 readings to measure concentration.Use OD 260/280 reading to indicate purity.

Conclusion

As shown in Figure 7, described herein method can be used for from separation of whole blood high yield, high quality genomic dna.

Embodiment 5 use dissolving of two steps and combined techniques with genomic dna from the pig separation of whole blood

1. 200 μ l fresh pig blood are distributed in 1.2ml 96 well culture plates.

2. adding 2 volumes dissolving damping fluid (0.5%SDS, 1%-Triton-X-100,10mM EDTA, 20mM Tris pH 7.4) and 12 μ l, 20 μ g/ μ l Proteinase Ks and slight inclination mixes 5 times.

3. cultivated 10 minutes down at 37 ℃.

4. adding 0.5 volume binding buffer liquid (40%PEG1000,1.5 M NaCl, 0.29% solid magnetic bead) and slight inclination mixes 10 times.

5. be placed on magnetic sheet last 15 minute.

6. remove supernatant liquor and it is discarded.

With the bead resuspending in 800 μ l lavation buffer solutions (6 M urea, 14%PEG1000,1 M NaCl) away from magnet.

8. be placed on magnetic sheet last 10 minute.

9. abandoning supernatant, repeating step 7 and 8 is to carry out the washing second time.

10. on magnet, bead is washed 3 times, do not upset bead with each 800 μ l, 70% ethanol.

11. in the end after the washing, remove all trace ethanol.With bead on magnetic sheet dry 5 minutes.

12. will not contain in the water of nuclease and rest testing table last 2 minute in 1 volume away from the bead resuspending of magnet, subsequently with bead resuspending once more.

13. be placed on magnetic sheet last 10 minute.

14. will contain the supernatant liquor of DNA transfers in the lancing door and stores.

The result

To be loaded on the 0.8% agarose e-gel from the 10 μ l DNA in 9 holes.Use OD 260 readings to measure concentration and output.Use OD 260/280 reading to indicate purity.

Conclusion

As shown in Figure 8, described herein method can be used for from separation of whole blood high yield, high quality genomic dna.

Embodiment 6 use dissolving of two steps and combined techniques with genomic dna from people's separation of whole blood

1. will be distributed in 1.2ml 96 well culture plates from the freezing human blood of 200 μ l of three different healthy donors.

2. adding 2 volumes dissolving damping fluid (0.5%SDS, 1%-Triton-X-100,10mM EDTA, 20mM Tris pH 7.4) and 12 μ l, 20 μ g/ μ l Proteinase Ks and slight inclination mixes 5 times.

3. cultivated 10 minutes down at 37 ℃.

4. adding 0.5 volume binding buffer liquid (40%PEG1000,1.5 M NaCl, 0.29% solid magnetic bead) and slight inclination mixes 10 times.

5. be placed on magnetic sheet last 15 minute.

6. remove supernatant liquor and it is discarded.

With the bead resuspending in 800 μ l lavation buffer solutions (6 M urea, 14%PEG1000,1 M NaCl) away from magnet.

8. be placed on magnetic sheet last 10 minute.

9. abandoning supernatant, repeating step 7 and 8 is to carry out the washing second time.

10. on magnet, bead is washed 3 times, do not upset bead with each 800 μ l, 70% ethanol.

11. in the end after the washing, remove all trace ethanol.With bead on magnetic sheet dry 5 minutes.

12. will not contain in the water of nuclease and rest testing table last 2 minute in 1 volume away from the bead resuspending of magnet, subsequently with bead resuspending once more.

13. be placed on magnetic sheet last 10 minute.

14. will contain the supernatant liquor of DNA transfers in the lancing door and stores.

The result

To be loaded on the 0.8% agarose e-gel from 10 μ l DNA of various kinds product.Use OD 260 readings to measure concentration and output.Use OD 260/280 reading to indicate purity.

Conclusion

As shown in Figure 9, described herein method can be used for from people's separation of whole blood high yield, high quality genomic dna.

Embodiment 7 total RNA separate (single step process) with culturing cell

Program:

1. (the every hole about 1 * 10, each hole from 96 well culture plates of the 293T cell cultivated ⁵Individual cell) removes all cells substratum in fully.To dissolve damping fluid (20mM Trisodium Citrate pH 7.5,10mMEDTA pH 8,1mM aurin tricarboxylic acid (Aurin tricarboxylic acid), 1%triton-x-100,2% sodium lauryl sarcosinate, 1 M LiCl, 0.075% solid magnetic bead) directly is drawn on the cell in each hole with dropper.Draw and mix by repeating dropper, till as if most cells left culture plate and entered in the suspension.It was left standstill several minutes, and dropper is drawn repeatedly to guarantee that all cells leaves culture plate more subsequently.

2. transfer to and quietly be placed on 1.7ml in the magnetic field not in the Ai Bende pipe (eppendorftube) of qiagen rnase enzyme.Make it leave standstill 5 minutes with bead and solution separating.

With settled solution from managing slow sucking-off and discarding.

4. with 1ml-1.5ml 70% ethanol once with the bead washing.As if directly dropper is drawn on the magnetic beads and it was left standstill 5 minutes or up to supernatant liquor till the clarification.Careful sucking-off ethanol and do not upset bead.

5. the Ai Bende pipe is moved away from magnet and add 30 μ l deoxyribonuclease I solution (10mMTris pH 7.5,2.5mM MgCl ₂, 0.5mM CaCl ₂, 0.4U/ μ l deoxyribonuclease I).

6. draw with dropper, till bead is got back in the suspension, and make it leave standstill 15 minutes with dna digestion away from magnet.

7. after the deoxyribonuclease I cultivation is finished, in bead/deoxyribonuclease I mixture, add 150 μ l binding buffer liquid (20mM Trisodium Citrate pH 7.5,10mM EDTA pH 8,1mM aurin tricarboxylic acid, 1%triton-x-100,2% sodium lauryl sarcosinate, 1 M LiCl, 30% Virahol) and draw 5 times, up to thorough mixing with dropper.

8. be put back into pipe on the magnetic bases and it was left standstill 5 minutes.

9. careful sucking-off supernatant liquor.

10. add 150 μ l lavation buffer solutions (25mM Trisodium Citrate, 1 M Guanidinium hydrochloride, 1%Triton-x-100) and will be away from magnet resuspending bead.Be put back on the magnetic bases immediately and it left standstill 5 minutes or up to as if clarification of supernatant liquor.

11. careful sucking-off supernatant liquor.

12. with the other four kinds of washings repeated washings that are stored in 70% ethanol.1ml 70% ethanol is drawn in the Ai Bende pipe on magnetic bases with dropper, it was left standstill 1 minute and removes.

13. behind the last washing with alcohol liquid of sucking-off, make reaction tubes on magnetic bases air-dry 10 minutes.

14. by with the complete resuspending of bead in 40 μ l not in the water of qiagen rnase enzyme and with purified product from the bead wash-out.

15. it was left standstill 5 minutes away from magnet, is placed on magnetic bases last 5 minute subsequently.

16. purified RNA is transferred in the new culture plate.

17. will be loaded on the 0.8% agarose e-gel from 12 samples of each culture plate.Use OD 260 readings to measure concentration and output.Use OD 260/280 reading to indicate purity.

The result

To be loaded on the 0.8% agarose e-gel from the RNA in 8 holes of two row of 96 well culture plates.Fig. 8 shows high yield, complete ribosome-RNA(rRNA) bands of a spectrum, the indication high quality rna.Use OD 260 readings to measure concentration and output.Use OD 260/280 reading to indicate purity.

Conclusion

As shown in Figure 10, described herein method can be used for separating high yield, the total RNA of high quality from cultivating mammalian cell.

Embodiment 8 total RNA separate with solid tissue

1. in each hole of 96 holes dissolvings culture plate, add 400 μ l dissolving damping fluid (2 M guanidinium isothiocyanates, 200mM Trisodium Citrate, 1mM DTT, 1%triton-x-100,1.2mg/ml Proteinase K), metallic bead and various rat tissues of 10mg at the most.With transparent culture plate seal strip culture plate is sealed and is connected on the wortex device.Wortex device is set at height and with culture plate vortex 10 minutes with the tissue that homogenizes.

2. culture plate is shifted out and is placed down in the thermocycler 15 minutes at 50 ℃ from wortex device.

3. it was at room temperature cooled off 5 minutes.

4. 400 μ l binding buffer liquid (80% Virahol, 1% magnetic beads solid) are added in the lysate and and mix for 5 times by drawing with dropper.At room temperature cultivated 2 minutes.

5. transfer to 400 μ l association reaction things in the fresh 1.2ml culture plate and be placed on magnetic sheet last 5 minute.

6. remove supernatant liquor and will remain 400 μ l association reaction things and transfer to last 5 minute of 1.2ml culture plate on the magnetic sheet.

7. remove supernatant liquor fully.

8. shift out culture plate and wash and mix from magnetic sheet by drawing 5 times by add 600 μ l 70%EtOH to every hole.

9. culture plate is put back into magnetic sheet last 5 minute.

10. remove washing with alcohol liquid fully and make culture plate leave magnetic sheet.

11. add 100 μ l deoxyribonuclease solution (10mM Tris pH 7.5,2.5mM MgCl ₂, 0.5mM CaCl ₂, 0.4U/ μ l deoxyribonuclease I), mix for 5 times and cultivated 15 minutes down by drawing at 37 ℃.

12. add 550 μ l binding buffer liquid (1 M guanidinium isothiocyanate, 100mM Trisodium Citrate, 0.5mM DTT, 0.5%triton-x-100,40% Virahol) again, draw by dropper and mixed and at room temperature cultivate 2 minutes for 5 times.

13. be placed on culture plate on the magnetic sheet and cultivated 15 minutes.

14. remove supernatant liquor and by adding 600 μ l 70%EtOH washing and mixing (culture plate on the magnet) by drawing 5 times.

15. step 13 is repeated four times again, washs altogether 4 times.

16. remove last washing with alcohol liquid fully and made bead at room temperature air-dry 10 minutes.

17. with culture plate from magnetic sheet shift out and by add minimum 40 μ l not the water of qiagen rnase enzyme come eluted rna and by draw mixing 10 times with dropper.At room temperature cultivated 2 minutes.

18. culture plate be put back into last 1 minute of magnetic sheet and carefully shift out through eluted rna.

The result

Analyze each RNA sample of 1 μ l by on Bioanalyzer 2100, carrying out capillary electrophoresis.Use OD 260 readings to measure concentration and output.Use OD 260/280 reading to indicate purity.

Conclusion

Figure 11 shows that described method can be used for separating the total RNA of high quality from solid tissue herein.

Embodiment 9 total RNA separate with whole blood

1. in 3 50mL centrifuge tubes 3 independent 10mL Paxgene pipes being collected blood sample mixes with 3.3mL dissolving damping fluid (4 M GITC, 400mM Trisodium Citrate, 2%TX-100), 13.2 μ l, 0.5 M DTT and 660 μ l Proteinase Ks (20mg/mL).

2. cultivated 30 minutes down at 55 ℃.

3. 6.2mL Virahol and 100 μ l, 5% solid magnetic bead are added in the sample.Thorough mixing.At room temperature cultivated 10 minutes.

4. centrifuge tube is placed on magnetic bases last 30 minute with bead and solution magnetic resolution.Carefully shift out supernatant liquor.

5. use 30mL washing soln (30% Virahol, 2 M GITC, 200mM Trisodium Citrate, 1%TX-100,1mM DTT) with the bead washed twice.

6. use 30mL 70% ethanol with the bead washed twice.

7. with air-dry 10 minutes of bead.

8. the bead resuspending, is transferred in the 2mL centrifuge tube not in the water of qiagen rnase enzyme in 640 μ l.At room temperature cultivated 5 minutes.

With bead on magnetic bases with the supernatant liquor magnetic resolution.Supernatant liquor is transferred in the 2mL pipe.

10. with 160 μ l deoxyribonuclease I damping fluid (10mM Tris pH 7.5,2.5mM MgCl ₂, 0.5mM CaCl ₂, 0.4U/ μ l deoxyribonuclease I) add in the sample.By drawing mixing up and down with dropper.

11. at room temperature cultivated 15 minutes.

12. add 540 μ l Virahols, 350 μ l dissolving damping fluid and the fresh bead of 10 μ l.Thorough mixing and at room temperature cultivating 5 minutes.

13. with bead and supernatant liquor magnetic resolution.Abandoning supernatant.

14. bead is washed 3 times with 1.6mL 70% ethanol.

15. with air-dry 10 minutes of bead.

16. with the bead resuspending in 50 μ l not in the water of qiagen rnase enzyme.At room temperature cultivated 5 minutes.

17. collect RNA and be stored under-80 ℃.

The result

Analyze each RNA sample of 1 μ l by on Bioanalyzer 2100, carrying out capillary electrophoresis.Use OD 260 readings to measure concentration and output.Use OD 260/280 reading to indicate purity.Be also shown in a sample of being analyzed on the 0.8% agarose e-gel.

Conclusion

Figure 12 shows that described method can be used for from the total RNA of separation of whole blood high quality herein.

Embodiment 10 uses to gargle and collects genomic dna and cheek cellular segregation

1. use 10ml mouth wash shua is gargled and is collected in the 50ml conical tube.

Under 3000 * g centrifugal 10 minutes with the cell granulation.

Abandoning supernatant and with the cell resuspending in 400 μ l resuspending damping fluids (10mM Tris pH 8,1mM EDTA pH 8,1.75ng/ μ l ribonuclease A).

4. add 600 μ l dissolving damping fluid (10mM Trsi pH 8,4 M Guanidinium hydrochlorides, 10%Tween-20,1% positive lauryl sarkosine) and mixing gently.

5. at room temperature cultivated 30 minutes.

6. 150 μ l lysates are transferred in the new pipe and merged, tilt to mix 10 times and be placed on the magnet with bead isolation with 150 μ l binding buffer liquid (18%PEG8000,2.2 M NaCl, 0.115% solid magnetic bead).

7. abandoning supernatant and with 200 μ l, 70% washing with alcohol three times.

8. with dry 5 minutes of bead.

9. add 40 μ l water, inclination mixes 10 times and is placed on the magnet to separate.

10. shift out the supernatant liquor that contains DNA.

The result

By coming the analytical separation genomic dna at the enterprising row agarose gel electrophoresis of 0.8%e-gel.The mean yield of every preparation is 1.8 μ g, but significantly changes between Different Individual.One 1 each sample of μ l and human ADP ribosylation factor 1 primer one are used among the PCR, obtain the 543bp product.

Conclusion

Figure 13 shows that described method can be used for use and gargles the collection scheme from cheek cellular segregation high quality, high yield gDNA herein.GDNA is used for follow-up pcr analysis.

Embodiment 11 uses wiping to collect genomic dna and cheek cellular segregation

1. with inner 20 times of aseptic swab wiping cheek.

2. swab is placed in the 200 μ l resuspending damping fluids (10mM Tris pH 8,1mM EDTA pH 8,1.75ng/ μ l ribonuclease A) and rotates 10 times to discharge cell.

3. add 300 μ l dissolving damping fluid (10mM Tris pH 8,4 M Guanidinium hydrochlorides, 10%Tween-20,1% positive lauryl sarkosine) and swab is rotated 10 times to mix.Swab is managed the bottom relatively to be pushed to discharge any liquid in fiber.

4. at room temperature cultivated 15 minutes.

5. 150 μ l lysates are transferred in the new pipe and merged, tilt to mix 10 times and be placed on the magnet with bead isolation with 150 μ l binding buffer liquid (18%PEG8000,2.2 M NaCl, 0.115% solid magnetic bead).

6. abandoning supernatant and with 200 μ l, 70% washing with alcohol 3 times.

7. with dry 5 minutes of bead.

8. add 40 μ l water, inclination mixes 10 times and is placed on the magnet to separate.

9. shift out the supernatant liquor that contains DNA.

The result

By come the genomic dna of analytical separation at the enterprising row agarose gel electrophoresis of 0.8%e-gel.The mean yield of every preparation is 1.8 μ g, but significantly changes between Different Individual.One 1 each sample of μ l and human cell's skeleton beta-actin primer are used for PCR, obtain 370 bp products.

Conclusion

Figure 14 shows that described method can be used for using the wiping scheme of collecting to separate high quality, high yield gDNA from the cheek cell herein.GDNA is used for follow-up pcr analysis.

The separation and the detection of embodiment 12 virus genom DNAs

1. 147 μ l dissolving damping fluid (4 M GITC, 400 mM Trisodium Citrates, 2%TX-100), 0.5 μ l PolyA RNA (10ug/ μ l) and 40 μ l Proteinase Ks (20mg/mL) are added in the 400 μ l human plasmas of the HBV that contains every milliliter of 50 copies.Mix at least 15 times by drawing up and down with dropper.

Under 56 ℃ with sample cultivation 30 minutes.

3. make the culture plate cool to room temperature.Add 260 μ l, 100% Virahol and 10 μ l, 5% solid magnetic bead.Mix at least 15 times by drawing up and down with dropper.

4. half sample (about 430 μ l) is transferred in the new hole.Culture plate was left standstill on testing table 5 minutes.

5. culture plate is placed on the magnetic sheet at least 7 minutes to separate.

6. sucking-off supernatant liquor.

7. culture plate is shifted out from magnet.Add 500 μ l lavation buffer solutions (30% Virahol, 2 M GITC, 200mM Trisodium Citrate, 1%TX-100,1mM DTT) and draw mixing 10 times with dropper.

8. culture plate was left standstill on testing table 5 minutes.Culture plate is put back into magnet last 7 minute.The sucking-off supernatant liquor.

9. washing step 7-8 is repeated twice again.

10. culture plate is shifted out from magnet.Add 500 μ l, 70% ethanol and draw mixing with the bead resuspending by dropper.

11. culture plate is put back into magnet last 7 minute.The sucking-off supernatant liquor.

12. 10-11 repeats twice again with the washing with alcohol step.Removing ethanol as much as possible after the washing for the third time.

13. make culture plate on magnet, leave standstill 20 minutes with dry bead.

14. make culture plate leave magnet.Add the not water of qiagen rnase enzyme of 20 μ l, dropper is drawn mixing and was cultivated 5 minutes.

15. culture plate is put back on the magnet.Supernatant liquor is transferred in the cleaning pipe.

The result

In 50 μ l PCR reaction, use 10 each sample of μ l and HBV Auele Specific Primers.The simulation extract of blood plasma that does not contain HBV is negative, and all 16 samples that contain HBV are positive.

Conclusion

Figure 15 shows that described method can be used for separating and the detection genomic dna from the virus of utmost point low copy number herein.

Embodiment 13 is from paraffin embedding sample separation genomic dna

Tissue digestion:

1. from 2mg-20mg paraffin-embedded tissue piece, tissue is cut to thin (10 μ m) cuts into slices and be placed in the 1.5ml pipe.Cover lid and pat pipe on testing table is fallen the pipe bottom up to tissue sample.

2. adding 100 μ l removes crosslinked damping fluid (50mM HEPES pH of buffer 7.9,1% ammonium hydroxide) and descends cultivation 1 hour at 70 ℃.

3. add 100 μ l dissolving damping fluids (50mM HEPES pH 7.9,1%SDS, 1mM EDTA) and 20 μ l 20mg/ml Proteinase Ks.

4. digested tissue 1 hour down at 55 ℃.

DNA extraction:

1. in the pipe that contains 200 μ l digestion tissue, add binding buffer liquid (200 μ l, 4 M GITC, 400mM Trisodium Citrate (pH 7.0), 2%Triton X-100; 6 μ l, 5% solid magnetic bead; 200 μ l Virahols).

2. mix and at room temperature cultivated 1 minute.

3. pipe is placed on magnetic bases last 1 minute.

4. abandoning supernatant.

With the bead resuspending in 300 μ l lavation buffer solutions (2 M GITC, 200mM Trisodium Citrate, 1%TX-100 and 30% Virahol), at room temperature cultivated 1 minute.

6. be placed on magnetic bases last 1 minute.Abandoning supernatant.

7. repeating step 6 and step 7.

With the bead resuspending in 200 μ l, 70% ethanol.At room temperature cultivated 1 minute.

9. abandoning supernatant.

10. step 9 and step 10 are repeated twice.

11. with the air-dry 10-15 of bead minute.

12. the bead resuspending in 50 μ l water, was at room temperature left standstill 1 minute.

13. pipe is placed on magnetic bases last 1 minute.

14. the DNA eluent is transferred in the new pipe.

The result

20 μ l DNA are loaded on 0.8% sepharose.DNA output from the 20mg tissue is 1.5-3 μ g.(going up gel) swimming lane 1 and 2 shows the typical pattern of the DNA of the separate tissue from be fixed in paraffin.(following gel) is from two gene specific pcr amplifications that extract sample (swimming lane 1 and 2), no template contrast (swimming lane 3) and positive control DNA (swimming lane 4).

Conclusion

As shown in Figure 16, described herein method can be used for genomic dna from the enough quality of paraffin-embedded tissue slice separation to be used for follow-up pcr amplification.

Although the present invention carries out particular display and description about its preferred embodiment, be understood by those skilled in the art that and under the situation that does not depart from the category of containing by the claim of enclosing of the present invention, can make the change of various forms and details it.

Claims (33)

1. the method for an isolated cell or organic genomic nucleic acids, it comprises:

A) merge

I) solid phase carrier, on its surface in conjunction with the functional group of reversible bind nucleic acid,

Ii) cell or organism, and

Iii) reagent, wherein said reagent make described cell or the dissolving of described organism and described cell or described organic genomic nucleic acids be deposited on the described solid phase carrier;

Thereby produce combination;

B) this combination is remained on described cell or organism and dissolving and described cell or organic genomic nucleic acids and described solid phase carrier take place reversibly under the bonded condition, thereby generation has the solid phase carrier with described cell of solid phase carrier bonded or organic genomic nucleic acids; And

C) described solid phase carrier is separated from this combination;

Thereby separate described cell or organic described genomic nucleic acids.

2. method according to claim 1, it further comprises makes c) described solid phase carrier contact with the elution buffer that makes described cell or organic genomic nucleic acids from described solid phase carrier wash-out.

3. method according to claim 1, wherein said solid phase carrier are magnetic particle.

4. method according to claim 3, wherein said magnetic particle have bag by the surface, and wherein said bag is selected from by the carboxyl bag by surperficial and amido bag by in the group of surface composition by the surface.

5. method according to claim 1, wherein said reagent comprises alcohol.

6. method according to claim 5, wherein said alcohol is selected from the group of being made up of ethanol, Virahol and polyalkylene glycol.

7. method according to claim 1, wherein said reagent further comprises salt.

8. method according to claim 7, wherein said salt is selected from by NaCl, LiCl and MgCl ₂In the group of being formed.

9. method according to claim 2, wherein elution buffer comprises water.

10. method according to claim 1, wherein said solid phase carrier use the method that is selected from the group of being made up of following method to separate from combination: apply magnetic field, apply vacuum filtration and apply centrifugal.

11. method according to claim 1, wherein said cell are selected from the group of being made up of bacterial cell, hemocyte, culturing cell, cheek cell and their combination; And described organism is a virus.

12. method according to claim 11, wherein said virus are hepatitis virus.

13. method according to claim 1, wherein said genomic nucleic acids are DNA or RNA.

Contact with reagent in being selected from the group of forming by following each reagent place 14. method according to claim 1, wherein said method further comprise the described solid phase carrier that makes in the step b): remove or the reagent of dna digestion, remove or digest RNA reagent, remove or the reagent of digesting protein and their combination.

15. the method for an isolated cell or organic genomic nucleic acids, it comprises:

A) merge cell or organism and make described cell or organism dissolved reagent, thereby produce first combination;

B) described first combination is remained under described cell or the organism generation dissolved condition, thereby produce lysate;

C) merge described lysate and binding buffer liquid, described binding buffer liquid comprises solid phase carrier, on this surface of solid phase carriers in conjunction with the functional group of reversible bind nucleic acid; And reagent, this reagent makes described cell or organic genomic nucleic acids be deposited on the described solid phase carrier, thereby produces second combination;

D) described second combination is remained under the reversible bonded condition of described cell or organic genomic nucleic acids and described solid phase carrier, thereby produce the solid phase carrier that has with described cell of solid phase carrier bonded or organic genomic nucleic acids;

E) described solid phase carrier is separated from described second combination; And

Thereby separate described cell or organic genomic nucleic acids.

16. method according to claim 15, it further comprises makes d) described solid phase carrier contact with elution buffer, described elution buffer makes described cell or organic genomic nucleic acids from described solid phase carrier wash-out.

17. method according to claim 15, wherein said magnetic particle have bag by the surface, wherein said bag is selected from the group of being made up of by the surface by surface and amido bag the carboxyl bag by the surface.

18. method according to claim 15, wherein said binding buffer liquid comprises alcohol.

19. method according to claim 18, wherein said alcohol is selected from the group of being made up of ethanol, Virahol and polyalkylene glycol.

20. method according to claim 15, wherein said binding buffer liquid further comprises salt.

21. method according to claim 20, wherein said salt is selected from by NaCl, LiCl and MgCl ₂In the group of being formed.

22. method according to claim 15, wherein said elution buffer comprises water.

23. method according to claim 15, wherein said magnetic particle use the method that is selected from the group of being made up of following method to separate from described combination: apply magnetic field, apply vacuum filtration and apply centrifugal.

24. method according to claim 15, wherein said cell are selected from the group of being made up of bacterial cell, hemocyte, culturing cell, cheek cell and their combination; And described organism is a virus.

25. method according to claim 24, wherein said virus are hepatitis virus.

Contact with reagent in being selected from the group of forming by following each reagent place 26. method according to claim 15, wherein said method further comprise the described solid phase carrier that makes in the step b): remove or the reagent of dna digestion, remove or digest RNA reagent, remove or the reagent of digesting protein and their combination.

27. a test kit, it comprises dissolving damping fluid, binding buffer liquid, the reagent that removes impurity and lavation buffer solution.

28. test kit according to claim 27, wherein said dissolving damping fluid comprises sodium lauryl sulphate, Triton X-100, EDTA and Tris-HCl.

29. test kit according to claim 27, wherein said binding buffer liquid comprises magnetic particle, polyoxyethylene glycol and sodium iodide.

30. test kit according to claim 27, the wherein said reagent that removes impurity is the reagent of digesting protein.

31. test kit according to claim 30, the reagent of wherein said digesting protein are Proteinase K.

32. test kit according to claim 27, wherein said lavation buffer solution comprises polyoxyethylene glycol and urea.

33. a test kit, it comprises:

A) dissolving damping fluid, it comprises sodium lauryl sulphate, Triton X-100, EDTA and Tris;

B) binding buffer liquid, it comprises magnetic particle, polyoxyethylene glycol and sodium iodide;

C) Proteinase K; With

D) lavation buffer solution, it comprises polyoxyethylene glycol and urea.

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