+

WO1998012351A1 - Procede de purification et eventuellement d'analyse d'acides nucleiques contenus dans des echantillons biologiques - Google Patents

Procede de purification et eventuellement d'analyse d'acides nucleiques contenus dans des echantillons biologiques Download PDF

Info

Publication number
WO1998012351A1
WO1998012351A1 PCT/EP1997/005129 EP9705129W WO9812351A1 WO 1998012351 A1 WO1998012351 A1 WO 1998012351A1 EP 9705129 W EP9705129 W EP 9705129W WO 9812351 A1 WO9812351 A1 WO 9812351A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
dna
synthetic resin
resin
subsequent
Prior art date
Application number
PCT/EP1997/005129
Other languages
German (de)
English (en)
Inventor
W. Kurt Roth
Dorothee Waschk
Stefan Zeuzem
Original Assignee
Roth W Kurt
Dorothee Waschk
Stefan Zeuzem
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE19638362A external-priority patent/DE19638362C1/de
Priority claimed from DE19731670A external-priority patent/DE19731670C2/de
Application filed by Roth W Kurt, Dorothee Waschk, Stefan Zeuzem filed Critical Roth W Kurt
Priority to AU47754/97A priority Critical patent/AU4775497A/en
Publication of WO1998012351A1 publication Critical patent/WO1998012351A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to a method for the purification and optionally analysis of nucleic acids from biological samples.
  • DNA in particular serves as the starting material for genetic analyzes in laboratory diagnostic research and in routine use.
  • nucleic acids such as DNA and RNA
  • biological samples in particular from samples of the human body, such as blood, body secretions, tissue samples, urine, stool and the like. the like
  • samples of the human body such as blood, body secretions, tissue samples, urine, stool and the like. the like.
  • the subsequent use in genetic analyzes is of particular importance, especially with regard to screening in tumor diagnosis and for the diagnosis of infectious agents such as viruses or bacteria.
  • the analysis of DNA which comes from exfoliated intestinal epithelial cells from stool samples, is of particular interest for the diagnosis of colorectal tumors. Isolation of the nucleic acid from whole blood is also of great interest in order to make the isolated nucleic acid available for genetic analysis.
  • the resulting DNA should be in high purity and be able to be subjected directly to the required subsequent reactions.
  • Nucleic acid diagnostics using DNA-a-amplification approaches in particular the polymerase chain reaction (polymerase chain reaction, hereinafter abbreviated to PCR) (see elfaiki, R. r Gelfand, DH, Stoffel, S., Scharf, SJ, Higuchi, R., Hörn, GT, Mullis, KB, Erlich, HA (1988), Science 239: 487-491), opens up diverse approaches to specific and at the same time sensitive DNA diagnostics, for example of tumors in the early stages that are not stressful and for screening are well suited. Due to the often small amount of DNA that can be isolated from a defined biological sample, such as stool samples, DNA amplification approaches such as the PCR technique seem to be a suitable method for the duplication of the DNA of interest.
  • the main difficulties are inhibitors which are isolated together with the nucleic acid of the biological sample when using conventional extraction methods and which inhibit the enzymes to be used in the nucleic acid amplification batches. It has been found that the DNA polymerase required for the PCR is inhibited. Stool samples and whole blood, in particular, are critical biological starting samples since they contain relatively large amounts of inhibitors.
  • the nucleic acid (DNA, RNA) resulting from the purification and isolation should be present in high purity and should be able to be subjected directly to the necessary subsequent reactions. The inhibitors must therefore be separated efficiently and selectively.
  • the DNA is usually isolated from cells. Cells are disrupted, for example, under strongly denaturing and possibly reducing conditions. The disruption of the cells with denaturing substances, e.g.
  • the DNA obtained by such a method e.g. from exfoliated intestinal epithelial cells from stool samples is only suitable to a limited extent for use in the subsequent subsequent reactions, in particular enzymatic amplification reactions such as PCR.
  • PCR enzymatic amplification reactions
  • Recent data show that only 103 cases out of a total of 230 extracted stool samples (efficiency of 44.7%) can be amplified using PCR (Villa, E., Dugani, A., Rebecchi, AM, Vignoli, A., Grottola, A., Buttafoco, P., Losi, L., Perini, M., Trande, P., Merighi, A., Lerose, R. and Manenti, F. (1996), Gastroenterology: 110: 1346-1353 ).
  • Intestinal epithelial cells are lysed and extracted with a buffer containing adsorbent (potato flour or potato starch or bovine serum albumin).
  • adsorbent potato flour or potato starch or bovine serum albumin.
  • the DNA-containing solution is used to break down proteins, for example nucleic acid-cleaving enzymes the proteinase K incubated.
  • the shortening of this process is due to the fact that the phenol extraction and subsequent ethanol precipitation are replaced by the use of centrifugation columns (QIAa p spin columns, QIAGEN GmbH). While the DNA reversibly attached to one
  • Binds silica membrane in the column disruptive compounds are pressed through the use of a suitable washing buffer due to the action of centrifugal forces and thus cleaned.
  • a suitable buffer By adding a suitable buffer, the purified DNA is eluted from the column as a result of a centrifugation step.
  • the object underlying the invention is therefore to provide an improved method for purifying or isolating nucleic acids (DNA, RNA and the like) from unpurified biological samples such as whole blood or stool samples; the nucleic acid should be present in sufficient quantity and purity so that it can be subjected to the necessary subsequent reactions.
  • the efficiency for a subsequent nucleic acid analysis reaction, in particular for amplification approaches such as PCR, is to be increased.
  • This object is achieved by a method for purification, optionally also analysis of nucleic acids from biological samples, the method comprising the step of reacting the nucleic acid-containing sample with an anion exchanger synthetic resin which has a binding affinity for
  • a characteristic of the present invention is the use of a bile acid binding anion exchange resin.
  • the binding affinity of the anion exchanger synthetic resin for bile acids which is preferably high, is a measure of the fact that the inhibitors for the nucleic acid analysis reaction which may have to be subsequently carried out, for example in the PCR reaction, are efficiently bound and thus separated while nucleic acids are not or are bound less and can therefore be easily recovered.
  • Another surprising result of the present invention is the very good selectivity of the anion exchange resin, which has the affinity for bile acids, for binding the inhibitors compared to binding the nucleic acid, which are not bound or are bound less in the presence of the inhibitors.
  • the nucleic acid is made available in high purity by the method according to the invention, typically in an A 2 6o / A 2e o ratio obtained via the absorption values at 260 nm and 280 nm of more than 1.5, in particular of more than 1.7.
  • the method according to the invention ensures adequate cleaning of inhibitors for any subsequent nucleic acid detection reactions while at the same time maintaining a sufficiently high nucleic acid concentration.
  • the binding affinity for bile acids characteristic of the synthetic resin used according to the invention, where typical bile acids such as cholic acid are suitable, is suitably in the range of an average binding capacity, as determined, for example, of cholic acid, of at least 0.1 equivalents of cholic acid per kg of dry synthetic resin (eq / kg).
  • the average binding capacity is preferably at least 0.5, more preferably at least 1.0 and in particular at least 2.0 equivalents of cholic acid per kg of dry synthetic resin.
  • the binding capacity to bile acids can be determined by reacting a defined amount of dry synthetic resin (50 g) with an appropriately concentrated bile acid solution (1% by weight aqueous solution of cholic acid) under suitable conditions with regard to pH and ionic strength (e.g. pH 6 , 3 at 0.9% by weight NaCl concentration). Cholic acid can be easily quantified (see for example JLIrvin et al. In "J.Biol.Chem.”, 153, 439 (1944)).
  • the total chloride exchange capacity of the synthetic resin used according to the invention is preferably more than 4 eg / kg. However, it must be taken into account that the total number of active, available anion exchange sites is not the actually decisive criterion for the effective binding of the inhibitors.
  • the characteristic underlying the nature of the bile acid-binding synthetic resin plays a further specific role here.
  • the quality of the binding or separation of the inhibitors can be determined indirectly by determining the efficiency of a subsequent nucleic acid analysis reaction.
  • a binding or separation of the inhibitors is achieved when an increase in efficiency can be determined in the subsequent nucleic acid analysis reaction, the efficiency before the cleaning according to the invention, in particular in the case of inhibitors heavily contaminated samples, may be zero.
  • the efficiency due to the different quantity and heterogeneity of the inhibitors depending on the biological sample, it can be assumed that the same biological sample was examined.
  • the efficiency is defined as the percentage of the specific PCR products to be determined in a PCR analysis approach, a simple PCR reaction, but advantageously using carrier protein, is carried out, for example according to the examples below.
  • the anion exchange resin used according to the invention binds the inhibitors from the biological starting material more selectively and more effective, so that the subsequent nucleic acid analysis reactions yield very high amplification rates even when using a simple PCR. According to the invention, significantly better results are obtained, even when using a smaller amount of anion exchange resin, and furthermore a smaller number of extraction steps are required; one incubation step is usually sufficient.
  • a comparison of conventional DNA purification and isolation methods was recently published by A. Kramvis et al. described in "Journal of Clinical Microbiology", 34, 2731-2733 (1996).
  • the selective separation of the inhibitors that can be achieved with the invention is also achieved with unpurified starting samples.
  • the method according to the invention has proven to be particularly effective in the case of starting samples which have been very problematic to date, e.g. in whole blood that has been treated with citrate and EDTA or the presumably PCR-inhibiting substance heparin, as well as in stool samples that come from the lysis of intestinal epithelial cells that have been exfoliated in stool and that have a high proportion of unknown inhibitors.
  • the advantages of the invention can also be achieved when applied to any other biological samples, for example other samples from the human body, such as body secretions, tissue samples, urine and the like. the like
  • the anion exchanger synthetic resin used according to the invention is a basic, in particular strongly basic polymer material of high molecular weight (for example in
  • 10 • 10 6 in particular 1 to 5 • 10 6 ), which is generally composed of an organic polymer or copolymer modified with tertiary and / or quaternary ammonium groups and whose characteristic feature is the affinity for bile acids described above.
  • the resin polymer is formed in a particularly suitable manner from a polystyrene which is crosslinked by divinylbenzene, preferably in a proportion of 0.5 to 5% by weight and in particular 1-3% by weight of divinylbenzene and in its network structure quaternary .Aitraionium groups, suitably in the form of benzyltrimethylammonium groups, are incorporated.
  • Colestyramine is the international free name for the copolymers of styrene (vinylbenzene) and about 2% divinylbenzene with the quaternary ammonium groups inserted into the network structure.
  • the colestyramine granules are known in the medical-therapeutic field as a highly hydrophilic, water-soluble, basic anion exchange resin for binding bile acids in bile acid loss syndromes and as a lipid-lowering agent for the treatment of hypercholesterolemia.
  • the lipid-lowering agent colestyramine is sold by STADApharm.
  • Crosslinked polyalkylamine anion exchange resins form further examples of a type of synthetic resin which is fundamentally suitable for use in the present invention.
  • the crosslinked polyalkylamine anion exchange resin is generally formed as a copolymer of a poly (lower) alkyl polyamine, for example diethylene triamine or tertaethylene pentamine, with epichlorohydrin (1-chloro-2, 3-epoxypropane).
  • An example of an anion-exchange synthetic resin of this type of polymer, which has a high affinity for bile acids and which has also achieved excellent results when used, is colestipol (average binding capacity of colestypol versus cholic acid 1.1 equivalents per kg resin dry weight) .
  • Colestipol is the international free name for the serum cholesterol-lowering copolymers of diethylene triamine and epichlorohydrin.
  • the Colestipol hydrochloride granules are also known per se as a water-insoluble, basic .anion exchange resin for binding bile acids in bile congestion and hypercholesterolemia (lipid-lowering agents). Colestipol hydrochloride was developed by Upjohn and was launched in 1977.
  • the cleaning efficiency compared to the inhibitors when using colestyramine is further increased compared to colestipol.
  • the polymers which form the synthetic resin also include other polymers and copolymers and the use of substituted representatives of the monomers which form the copolymers, provided that the inhibitors which may be present, in analogy with the ability to bind to bile acids, by increasing the efficiency of the subsequent nucleic acid analysis reaction can be determined and separated.
  • Anion exchange resins contain other additives known per se in conventional amounts, e.g. Superplasticizer.
  • the anion exchange resin also contains suitable salt partners for charge saturation; the tertiary ammonium groups are suitably in the hydrochloride form, while quaternary ammonium groups are suitably associated with chloride.
  • the resin to be used can first be in a suitable dry form, for example in the form of granules, or it can already be in an aqueous form
  • the resin suspension used for the reaction is usually buffered, suitably in the range from slightly acidic (approximately pH 5-6) to weakly basic (approximately pH 9-10).
  • nucleic acid types in particular DNA and RNA, are suitable as nucleic acid. Because of its greater importance, but above all because nucleic acid amplification reactions are usually based on DNA samples (cf. PCR), the invention is further described below, primarily for the purification or isolation of DNA.
  • the cells In the case of biological starting samples in which the nucleic acid to be isolated is present intracellularly, such as stool samples containing exfoliated intestinal epithelial cells, the cells must first be lysed in order to disrupt the intracellular material.
  • the lysis or disruption of the cells can be achieved by a simultaneous physical and chemical action on the sample containing the body cells.
  • the sample material can be frozen (-80 ° C) before lysis.
  • the lysis buffer suitably contains a denaturing agent, for example sodium dodecyl sulfate (SDS) or other detergents, which brings about chemical digestion of the cells, while stirring the suspension, for example with an automatic, mechanical stirring system, favors mechanical digestion.
  • SDS sodium dodecyl sulfate
  • a centrifugation step can remove cell debris, undigested food residues or other macro residues.
  • the nucleic acid-containing cell lysate is treated with the special anion-exchange resin used according to the invention.
  • the resin in this case can be suitably pre-swelled in the lysis buffer.
  • the amount of the special synthetic resin used is preferably 10% by weight and less, based on the total weight of the treated sample. Above this amount there may be a tendency that not only the inhibitors but also the desired nucleic acid is increasingly bound to the resin.
  • aqueous, up to 10% by weight, in particular 5 to 10% by weight, suspension of the synthetic resin binds the inhibitors in sufficient quantity and at the same time does not, or only insignificantly, reduce the amount of DNA present in the aqueous solution.
  • the amount of synthetic resin used is inversely related to the frequency of the reaction. That is, with a relatively high salary, in particular with 7.5 to 10% by weight of resin suspension in an aqueous medium, a single reaction is preferable, while in the medium content range, approximately from 2.5 to 7.5% by weight and in particular around 5% by weight ( ⁇ 1 wt.), A two or more times implementation delivers better results.
  • the preferred ratio of resin content to frequency of implementation also depends on the sample material to be examined. For stool samples, a single reaction with 10% by weight or a double reaction with 5% by weight of copolymer resin is particularly suitable. In the case of whole blood, content ranges below 5% by weight are preferred, two reactions with 2.5% by weight of copolymer resin being particularly suitable.
  • the easiest way is to add the resin suspension to the nucleic acid-containing (possibly not pre-cleaned) sample, mix it very well, and then separate it again from the copolymer resin.
  • the latter can be conveniently carried out by centrifuging off the resin granules.
  • the reaction can also be carried out by means of a separation using a chromatography system using the special anion exchange resin.
  • the DNA can be further purified and isolated. It is advantageous to use non-specific proteinases such as Proteinase K to break down proteins and nucleic acid-cleaving enzymes. Then you get a viscous, gelatinous liquid. From this, the DNA is isolated from the aqueous phase, preferably by means of phenol extraction and subsequent ethanol precipitation. Alternatively, the DNA can be removed by using a detergent, for example a chaotropic guanidine-containing detergent (such as DNAzol TM from Gibco BRL), followed by ethanol precipitation from the aqueous phase isolate.
  • a detergent for example a chaotropic guanidine-containing detergent (such as DNAzol TM from Gibco BRL), followed by ethanol precipitation from the aqueous phase isolate.
  • the DNA-containing solution digested with Proteinase K is mixed with the detergent and ethanol and immediately pelleted by a centrifugation step. Since neither organic solvents (phenol, chloroform) are used, nor are several centrifugation steps required for extraction, this process is very user-friendly and time-saving.
  • organic solvents such as phenol or chaotropic detergents for isolating DNA
  • centrifugation columns known for this purpose, for example QIAamp-spin columns TM from Qiagen TM.
  • the quality of the nucleic acid sample required for this is provided by the method according to the invention.
  • the expected .amplicate could even be obtained in up to 100% of the samples tested.
  • the DNA purified or isolated according to the purification method according to the invention is preferably subjected to a PCR which is carried out in the presence of a carrier protein, such as bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the carrier protein concentration is chosen to be high, preferably more than 50 ⁇ g / ml. Very good amplification rates have been found for carrier protein concentrations in the range of 120-200 ⁇ g / ml.
  • relatively high concentrations of nucleotides required for the PCR deoxyribonucleoside
  • Triphosphate Triphosphate
  • primers and DNA polymerases such as Tag DNA polymerase advantageous.
  • the nucleotide concentrations are preferably in the range of 150-225 ⁇ M.
  • primer concentration is in the range from 0.75 to 1.25 ⁇ M.
  • the content of DNA polymerase is suitably in the range of 2 to 3 units per 50 ul batch.
  • the amplification is preferably carried out by a simple PCR in which 30-35 temperature cycles are carried out.
  • 200 mg of stool material is deep-frozen for at least one hour at -80 ° C, then mixed with 600 ⁇ l lysis buffer (500 M Tris, 75 mM EDTA, 10 M NaCl, 1% SDS, pH 9.0) and homogenized.
  • the lysed sample is 10 min. Centrifuged at 4 ° C and 6000 xg in an Eppendorf table centrifuge to remove coarse stool particles, cell debris, bacteria and food residues.
  • the supernatant is a second time at 4 ° C, 20,000 xg 10 min. centrifuged.
  • the DNA-containing supernatant is mixed with the same volume of a colestyramine solution (5% colestyramine in lysis buffer), mixed well, 2 min.
  • the DNA can be pelleted from the aqueous, upper phase by ethanol precipitation, by adding 1/10 volume of 3 M sodium acetate, pH 5.2 and 2.5 times the volume of 100% ethanol.
  • the DNA pellet washed in 75% ethanol and dried at room temperature, is dissolved in 100 ⁇ l of distilled water.
  • the DNA yield is 10-15 ⁇ g per 200 mg stool sample with an A 260/2 eo ratio of 1.7. 5 ⁇ l of this DNA solution are used to amplify defined genes / gene segments in a 50 ⁇ l PCR approach used.
  • the amplification mixture is composed as follows:
  • BSA bovine serum albumin
  • 200 mg of stool material is frozen for at least one hour at - 80 ° C, then mixed with 600 ⁇ l lysis buffer (500 mM Tris, 75 mM EDTA, 10 mM NaCl, 1% SDS, pH 9.0) and homogenized.
  • the lysed sample is 10 min. Centrifuged at 4 ° C and 5000 xg in an Eppendorf table centrifuge to remove coarse stool particles, cell debris, bacteria and food residues.
  • the supernatant is a second time at 4 ° C, 13000 xg 10 min. centrifuged.
  • the DNA-containing supernatant is mixed with the same volume of a colestipol hydrochloride solution (10% colestipol hydrochloride in lysis buffer), mixed well, 2 min.
  • the clear supernatant is incubated with proteinase K (final concentration 100 ⁇ g / ml) for 2 hours at 56 ° C.
  • the digested sample is mixed with the same volume of a phenol-chloroform-isoamyl alcohol solution (25: 24: 1), mixed well and 5 min. centrifuged at room temperature and 13000 xg.
  • the aqueous, upper phase becomes a again with the same volume of a chloroform-isoamyl alcohol solution (24: 1) and centrifuged as described above.
  • the DNA can be removed from the aqueous, upper phase by ethanol precipitation, by adding 1/10 volume of 3 M sodium acetate, pH 5.2 and 2.5 times the volume
  • the DNA pellet washed in 75% ethanol and dried at room temperature, is dissolved in 100 ⁇ l of distilled water.
  • the DNA yield is 15-20 ⁇ g per 200 mg stool sample with an A 260 / A 280 ratio of 1.9. 5 ⁇ l of this DNA solution are used for the amplification of defined genes / gene segments in a 50 ⁇ l PCR approach.
  • the amplification mixture is identical to that of Example 1.
  • 500 ⁇ l citrate, heparin or EDTA blood or frozen and thawed blood are mixed with 500 ⁇ l lysis buffer (500 mM Tris, 75 mM EDTA, 10 mM NaCl, 1% SDS, pH 9.0) and mixed well.
  • 500 mM Tris, 75 mM EDTA, 10 mM NaCl, 1% SDS, pH 9.0 500 mM Tris, 75 mM EDTA, 10 mM NaCl, 1% SDS, pH 9.0
  • the DNA-containing solution is mixed with the same volume of a colestyramine solution (2.5% colestyramine in lysis buffer), mixed well, 2 min. at
  • Phenol added, mixed well and 5 min. centrifuged at room temperature and 20,000 xg.
  • the aqueous, upper phase is again mixed with the same volume of a chloroform-isoamyl alcohol solution (24: 1) and as described above centrifuged.
  • the DNA can be removed from the aqueous, upper phase by ethanol precipitation , by adding 1/10 volume of 3 M sodium acetate, pH 5.2 and 2.5 times the volume of 100% ethanol.
  • the DNA pellet washed in 75% ethanol and dried at room temperature, is dissolved in 30 ⁇ l of distilled water.
  • the DNA yield is 5-10 ⁇ g per 500 ⁇ l whole blood with an ⁇ 2 6o / 2 8o ratio of 1.7. 5 ⁇ l of this DNA solution are used for the amplification of defined genes / gene segments in a 50 ⁇ l PCR approach.
  • the amplification mixture is identical to that of Example 1.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de purification et éventuellement d'analyse d'acides nucléiques contenus dans des échantillons biologiques, ledit procédé consistant à faire réagir l'échantillon contenant l'acide nucléique avec une résine synthétique échangeuse d'anions, possédant une affinité de liaison, de préférence élevée, avec les acides biliaires. Les inhibiteurs éventuellement présents pour la réaction d'analyse éventuellement effectuée ensuite, sont liés à la résine échangeuse d'anions puis séparés.
PCT/EP1997/005129 1996-09-19 1997-09-18 Procede de purification et eventuellement d'analyse d'acides nucleiques contenus dans des echantillons biologiques WO1998012351A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47754/97A AU4775497A (en) 1996-09-19 1997-09-18 Method for purifying and eventually analyzing nucleic acids from biological test samples

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19638362A DE19638362C1 (de) 1996-09-19 1996-09-19 Verfahren zur Reinigung von Nukleinsäuren oder zur Isolierung von Nukleinsäuren aus körperzellenhaltigen Proben unter gleichzeitiger Abreinigung von PCR-Inhibitoren
DE19638362.5 1996-09-19
DE19731670.0 1997-07-23
DE19731670A DE19731670C2 (de) 1997-07-23 1997-07-23 Verfahren zur Reinigung und gegebenenfalls Analyse von Nukleinsäuren aus biologischen Proben

Publications (1)

Publication Number Publication Date
WO1998012351A1 true WO1998012351A1 (fr) 1998-03-26

Family

ID=26029568

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/005129 WO1998012351A1 (fr) 1996-09-19 1997-09-18 Procede de purification et eventuellement d'analyse d'acides nucleiques contenus dans des echantillons biologiques

Country Status (2)

Country Link
AU (1) AU4775497A (fr)
WO (1) WO1998012351A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0915171A3 (fr) * 1997-11-04 2000-03-22 Becton, Dickinson and Company Procédé de traitment d'un échantillon à l'aide d'un échangeur d'ions
WO2000071698A3 (fr) * 1999-04-30 2001-02-15 Connex Ges Zur Optimierung Von Melange pour neutraliser des inhibiteurs d'enzymes
WO2001010554A3 (fr) * 1999-08-09 2001-08-23 Bilatec Ges Zur Entwicklung Bi Robot de laboratoire, procede et trousse de reactifs permettant d'isoler des acides nucleiques
US7347976B2 (en) 2001-12-20 2008-03-25 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrix
US7871827B2 (en) 2001-12-20 2011-01-18 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using anion exchange
US7939249B2 (en) 2003-12-24 2011-05-10 3M Innovative Properties Company Methods for nucleic acid isolation and kits using a microfluidic device and concentration step
US7981600B2 (en) 2003-04-17 2011-07-19 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using an anion exchange material that includes a polyoxyalkylene
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2391730A1 (fr) * 1977-05-23 1978-12-22 Bristol Myers Co Composition a base d'une resine styrene-divinylbenzene d'echange anionique portant des groupes ammonium quaternaire, destinee a fixer les acides biliaires
EP0270017A2 (fr) * 1986-12-01 1988-06-08 Syngene, Inc. Procédé d'isolation et de purification d'acides nucléiques d'échantillons biologiques
DE4136322A1 (de) * 1991-11-05 1993-05-06 Hoechst Ag, 6230 Frankfurt, De Pektinderivate als adsorptionsmittel fuer gallensaeuren, mit gallensaeuren beladene pektinderivate und verfahren zu deren herstellung sowie deren anwendung als arzneimittel
JPH0623281A (ja) * 1992-07-10 1994-02-01 Babcock Hitachi Kk 液体クロマトグラフィー用陰イオン交換樹脂
JPH07277993A (ja) * 1994-04-01 1995-10-24 Sekisui Chem Co Ltd 胆汁酸吸着剤

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2391730A1 (fr) * 1977-05-23 1978-12-22 Bristol Myers Co Composition a base d'une resine styrene-divinylbenzene d'echange anionique portant des groupes ammonium quaternaire, destinee a fixer les acides biliaires
EP0270017A2 (fr) * 1986-12-01 1988-06-08 Syngene, Inc. Procédé d'isolation et de purification d'acides nucléiques d'échantillons biologiques
DE4136322A1 (de) * 1991-11-05 1993-05-06 Hoechst Ag, 6230 Frankfurt, De Pektinderivate als adsorptionsmittel fuer gallensaeuren, mit gallensaeuren beladene pektinderivate und verfahren zu deren herstellung sowie deren anwendung als arzneimittel
JPH0623281A (ja) * 1992-07-10 1994-02-01 Babcock Hitachi Kk 液体クロマトグラフィー用陰イオン交換樹脂
JPH07277993A (ja) * 1994-04-01 1995-10-24 Sekisui Chem Co Ltd 胆汁酸吸着剤

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DEUTER R. ET AL.,: "A method for preparation of fecal DNA suitable for PCR", NUCLEIC AIDS RESEARCH, vol. 23, no. 18, - 25 September 1995 (1995-09-25), pages 3800 - 3801, XP002017414 *
KRAMVIS A. E AL.,: "Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, genereleaser, and the Phenol-chloroform method", J. CLIN. MICROBIOLOGY, vol. 34, no. 11, - November 1996 (1996-11-01), pages 2731 - 2733, XP002054107 *
PATENT ABSTRACTS OF JAPAN vol. 018, no. 233 (C - 1195) 28 April 1994 (1994-04-28) *
PATENT ABSTRACTS OF JAPAN vol. 096, no. 002 29 February 1996 (1996-02-29) *
WIDJOJOATMODJO M. N. ET AL.,: "The magnetic immuno polymerase chain raction assay for direct detetction of salmonellae in fecal samples", J. CLIN. MICROBIOLOGY, vol. 30, no. 12, - December 1992 (1992-12-01), pages 3195 - 3199, XP002054106 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0915171A3 (fr) * 1997-11-04 2000-03-22 Becton, Dickinson and Company Procédé de traitment d'un échantillon à l'aide d'un échangeur d'ions
US6241980B1 (en) 1997-11-04 2001-06-05 Becton, Dickinson And Company Sample processing method using ion exchange resin
US6265224B1 (en) 1997-11-04 2001-07-24 Becton, Dickinson And Company Sample processing method using ion exchange resin
WO2000071698A3 (fr) * 1999-04-30 2001-02-15 Connex Ges Zur Optimierung Von Melange pour neutraliser des inhibiteurs d'enzymes
WO2001010554A3 (fr) * 1999-08-09 2001-08-23 Bilatec Ges Zur Entwicklung Bi Robot de laboratoire, procede et trousse de reactifs permettant d'isoler des acides nucleiques
US7347976B2 (en) 2001-12-20 2008-03-25 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrix
US7871827B2 (en) 2001-12-20 2011-01-18 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using anion exchange
US11572581B2 (en) 2002-06-07 2023-02-07 DNA Genotek, Inc. Compositions and methods for obtaining nucleic acids from sputum
US7981600B2 (en) 2003-04-17 2011-07-19 3M Innovative Properties Company Methods and devices for removal of organic molecules from biological mixtures using an anion exchange material that includes a polyoxyalkylene
US7939249B2 (en) 2003-12-24 2011-05-10 3M Innovative Properties Company Methods for nucleic acid isolation and kits using a microfluidic device and concentration step
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11536632B2 (en) 2011-06-19 2022-12-27 DNA Genotek, Inc. Biological collection system
US11549870B2 (en) 2011-06-19 2023-01-10 DNA Genotek, Inc. Cell preserving solution
US11592368B2 (en) 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11198899B2 (en) 2014-03-07 2021-12-14 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples

Also Published As

Publication number Publication date
AU4775497A (en) 1998-04-14

Similar Documents

Publication Publication Date Title
DE69734263T2 (de) Verfahren zur Isolierung von Ribonucleinsäuren.
DE4321904B4 (de) Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen
DE69802191T3 (de) Festphasen-Nukleinsäure-Isolierung
EP1851313B1 (fr) Méthode pour l'isolation d'acides nucléiques dans laquelle les acides nucléiques sont immobilisés à haute température sur une matrice
DE3787445T2 (de) Verfahren zur Isolierung von langkettiger Nukleinsäure.
DE69432540T2 (de) Verfahren zur Herstellung von Nucleinsäuren aus einer biologischen Probe bei niedrigem pH und unter Verwendung einer Säureprotease
DE19856064C2 (de) Universelles Verfahren zur Isolierung von DNA aus beliebigen Ausgangsmaterialien
DE69730974T2 (de) Verfahren zur isolierung von nukleinsäuren unter anwendung von alkaliner phosphatase
EP1146049B1 (fr) Procédé de séparation de structures d'acides nucléiques à deux brins et à un brin
EP0765335B1 (fr) Procede universel d'isolement et de purification d'acides nucleiques a partir de quantites extremement reduites de differents materiaux de depart fortement contamines
DE10084502B4 (de) Verfahren zur Reinigung von Nukleinsäure mit Siliciumcarbid
DE69533317T2 (de) Isolierung von nukleinsäuren
DE602004009022T2 (de) Verfahren und Reagentien zur Extraktion von RNA und Verfahren zur Analyse von biologischem Material
DE19900638C2 (de) Methode zur Isolierung von DNA aus biologischen Materialien
DE60012549T2 (de) Verfahren zur isolierung von nukleinsäuren und kit
DE69636553T2 (de) Nukleinsäuren bindender magnetischer Träger und Verfahren für die Nukleinsäureisolierung unter dessen Verwendung
WO1999040098A1 (fr) Procede pour isoler et purifier des acides nucleiques
EP2501811A1 (fr) Procédé d'enrichissement et isolement sélectifs d'acides nucléiques microbiens et facultativement en outre viraux
WO2000024927A1 (fr) Procedes et moyens pour l'isolement et la purification d'acides nucleiques sur des surfaces
DE60014399T2 (de) Schnelle und wirksame dns-immobilisierung aus einer probe ohne die verwendung von zelllysereagenzien
WO2007060248A1 (fr) Procédé d'isolation d'acides nucléiques à partir de matériaux de départ quelconques
WO1998012351A1 (fr) Procede de purification et eventuellement d'analyse d'acides nucleiques contenus dans des echantillons biologiques
EP1560926B1 (fr) Nouvelles formulations de tampons pour isoler, purifier et recuperer des acides nucleiques a chaine longue et a chaine courte
DE69130394T2 (de) Nicht-ätzende zusammensetzung und verfahren zur gewinnung von nukleinsäuren
EP2010672B1 (fr) Formulations et procédé d'isolement d'acides nucléiques contenus dans des matières premières complexes quelconques et analyse génétique complexe consécutive

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV GH

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998514297

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载