WO1998012351A1 - Method for purifying and eventually analyzing nucleic acids from biological test samples - Google Patents

Method for purifying and eventually analyzing nucleic acids from biological test samples Download PDF

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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
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characterized
method according
nucleic acid
dna
method
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PCT/EP1997/005129
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German (de)
French (fr)
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W. Kurt Roth
Dorothee Waschk
Stefan Zeuzem
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Roth W Kurt
Dorothee Waschk
Stefan Zeuzem
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006Extracting 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
    • C12N15/101Extracting 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Abstract

The invention concerns a method for purifying and eventually analyzing nucleic acids from biological test samples. In said method the test sample containing nucleic acid is made to react with an anion-exchanging synthetic resin, which has a preferably high bonding affinity with bile acid. The possible existing inhibitors for the eventually ensuing nucleic acid analysis reaction are bonded and separated by the anion-exchanging synthetic resin.

Description

A process for cleaning and optionally Anal of nucleic acids from biological samples

description

The present invention relates to a method for cleaning and, where appropriate, analysis of nucleic acids from biological samples.

The purification of nucleic acids plays a central role in molecular biology. In particular, the DNA is used as a raw material for genetic analysis in the laboratory diagnostic research and in routine use.

The isolation of nucleic acids such as DNA and RNA from biological samples, in particular samples from the human body, such as blood, body secretions, tissue samples, urine, stool u. like. , For subsequent use in genetic analysis is of particular importance, especially with regard to screening in tumor diagnosis and for the diagnosis of infectious agents such as viruses or bacteria.

For example, the analysis of DNA derived from exfoliated epithelial cells from stool samples of particular interest for the diagnosis of colorectal tumors. Of great interest is the isolation of nucleic acid from whole blood to make it accessible to the isolated nucleic acid genetic Anal.

In particular, the DNA thereby obtained should be present in high purity and the required subsequent reactions can be subjected directly. AAinplifikationsansätzen a nucleic acid diagnostics using DNA, in particular the polymerase chain reaction (Polymerase Chain Reaction, hereinafter abbreviated to PCR) (see Fig. Ξaiki, R. r Gelfand, DH, Stoffel, S., Scharf, SJ, Higuchi, R. horn, GT, Mullis, KB, Erlich, HA (1988) Science 239: 487-491), opens various approaches to a specific, yet sensitive DNA diagnostics, eg of early stage tumors that are not onerous and screening are well suited. Due to the often low amount of DNA which can be from a defined biological sample, such as in stool samples, isolated DNA Amplifikationsansätze as the PCR technique appears to be a suitable method for copying the DNA of interest.

However, major problems are inhibitors which are in common with the nucleic acid of the biological sample isolated in the application of common extraction methods, and which inhibit inserted into the nucleic acid Amplifikationsansätzen enzymes. Thus, it has been found that the time required for the PCR DNA polymerase is inhibited. In particular, stool samples and whole blood are critical biological output samples, as they are subject to relatively large amounts of inhibitors. The purification and isolation by the resulting nucleic acid (DNA, RNA) is intended to be present in high purity and the required subsequent reactions can be subjected directly. The inhibitors must therefore be efficiently and selectively separated. Typically, the DNA is isolated from cells. In this case cells are disrupted, for example under strongly denaturing and, optionally, reducing conditions. Widely used is the disruption of the cells with denaturing substances, eg. As detergents, and the use of certain enzymes to .Abbau of proteins and nucleic acids. Thus, for example, sodium dodecyl sulphate (SDS) as denaturing agent, proteinase K to degrade proteins and RNase A for degradation of ribonucleic acids (RNA). For complete denaturation of proteins, the DNA containing solution is extracted with the organic solvent of phenol. By the subsequent ethanol precipitation, the concentration of the DNA and at the same time the removal of remaining traces of phenol from the deproteinized aqueous solution is carried out. (Maniatis, T., Fritsch, EF and Sa brook, S. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor University Press, Cold Spring Harbor).

The recovered DNA by such a method, for example, from exfoliated epithelial cells from stool samples, is only limited for use in the subsequent secondary reactions, particularly enzymatic amplification reactions such as PCR. To demonstrate more recent data indicate that only in cases 103 of a total of 230 fecal specimens extracted (efficiency of 44.7%), the DNA amplified by PCR can (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 ).

Deuter et al. published in 1995 a method for the isolation of DNA from stool samples, time shortens and simplifies the method described (Deuter, R., Pietsch, R., Hertel, S. and Muller, 0. (1995) Nucl. Acids Res., the, 23 : 3800 3801). The exfoliated be present in the stool

Intestinal epithelial cells are lysed and extracted with an adsorbent (potato flour or potato starch, or bovine serum albumin) containing buffer. For the degradation of proteins, for example, nucleic acid degrading enzymes, the DNA-containing solution is incubated with the proteinase K. The reduction in time of this method resides in that the phenol extraction followed by ethanol precipitation (QIAa p spin columns, Qiagen GmbH) are replaced by the use of centrifugation columns. While the DNA reversibly to a

Silica membrane binds in the column, are interfering compounds by using a suitable washing buffer by the action of centrifugal forces forced through the membrane and thus cleaned. By adding a suitable buffer, the purified DNA is eluted as a result of centrifugation of the column. However, because not fully allow liquids to remove from such membranes, one always has to reckon with a loss of DNA yield. The DNA obtained by this process is not sufficiently good quality (A 2 6o / o A 28 = 1) and quantity (2 ug

DNA / 200 mg of stool sample) before. Secondary reactions, in particular the PCR, require a high, reproducible yield of the crude preparations of DNA under simultaneous intensive cleaning interfering inhibitors. The amplification of a defined gene / gene segment of the after Deuter et al. Method described DNA prepared by a simple PCR shows an efficiency of 16%. Also by a subsequent phenol extraction of DNA, the amplification efficiency can be increased only by 16 to 40%. Only the use of a nested ( "nested") PCR, which is characterized by an increased sensitivity and sensitivity, while thinning potential inhibitors, distinguished (Newton, CR and Graham, A. (1994), PCR, Spektrum Akademischer Verlag Heidelberg, Federal Republic of Germany ), achieves an increased Amplifikationsausbeute of 66%.

It is desirable to isolate the DNA in sufficient quantity and in a reproducible good quality, so that defined genes / gene segments in a simple, ie non-nested PCR can be reproduced. The implementation of a nested PCR shows up for specific applications (eg. As routinemäße investigations in the diagnostic laboratory) adversely, since the rate of false positives is increased due to product contamination.

The object underlying the invention is thus to provide an improved process for the purification or isolation of nucleic acids (DNA, RNA and the like.) Of unpurified biological samples such as whole blood or fecal samples; it should be present in sufficient quantity and purity of the nucleic acid, so that they can be subjected to necessary subsequent reactions. The efficiency of .a subsequent nucleic acid analysis reaction, especially for Amplifikationsansätze such as PCR, is to be increased.

This object is achieved by a method for cleaning, possibly also analysis of nucleic acids from biological samples, which method comprises the step that the nucleic acid-containing sample is reacted with an anion exchange resin which has a binding affinity to

Bile acids, said inhibitors are tied for the optional subsequent nucleic acid .Analysereaktion to the anion exchange resin • and separated.

A charakertistisches feature of the present invention is the use of a bile acid-binding anion exchange resin.

The binding affinity of the anion exchange resin to bile acids, which is preferably high, is a scaling factor for the fact that the inhibitors of the optionally subsequently be performed nucleic acid Analysenereaktion, for example, in the PCR reaction, efficiently bound and thus be separated, while the nucleic acids do not or less be bound and thus can be easily recovered.

The reason why a .Anionenaustauscher resin very efficiently binds with high affinity for bile acids are inhibitors and separating, is not clear. It can be assumed, however, that - although bile acids themselves do not seem which are inhibitors of nucleic acid analysis of reactions - the bile acid structure with on the one hand permanently negative carboxyl charge group and the other relatively hydrophobic, are methylated in the ß-position of the steroid structural portion which however in turn, is usually derivatized by hydrophilic hydroxyl groups in α-position, obviously relatively well reflected in the the present inhibitors coarse structure distribution, so that an analog strong affinity for the inhibitors composed.

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 of the inhibitors compared to the binding of the nucleic acid that are not bound or less in the presence of the inhibitors. At the same time the nucleic acid in high purity is provided by the inventive method, typically in an obtained on the absorbance values at 260 nm and 280 nm ratio A 2 6o / A 2e o of more than 1.5, especially of above 1.7.

The inventive method a sufficient .Abreinigung of inhibitors for optional subsequent nucleic acid detection reactions is ensured while simultaneously retaining a high enough nucleic acid concentration.

The characteristic of the invention used resin bond affinity for bile acids, typical bile acids such as cholic acid come into consideration, is suitably in the range of a determined relative to, for example, cholic average binding capacity of at least 0, 1 equivalents of cholic acid per kg of dry resin (eq / kg). Preferably, the average binding capacity at least 0.5, more preferably at least 1.0 and especially at least 2.0 equivalents of cholic acid per kg of dry resin. The binding capacity for bile acids can be determined by reacting a defined amount of dry resin (50 g) with a reasonably concentrated bile acid solution (1 wt .-% term aqueous solution of cholic acid), under appropriate conditions related. PH and ionic strength (for example, pH 6 , 3 at 0.9 wt% -. NaCl concentration). Cholic acid can be easily quantified (see, for example JLIrvin et al., "J. Biol.", 153, 439 (1944)).

The total chloride exchange capacity of the resin used in the invention, as determined by conventional titration method is preferably more than 4 EC / kg. However, it is to be considered that the total number of active anion exchange sites available is not the actually relevant criterion for effective binding of the inhibitors. The underlying in the nature of bile acid-binding resin characteristics plays a more specific role.

The quality of the bond and separation of the inhibitors may be determined indirectly by determining the efficiency of a subsequent nucleic acid analysis reaction. A binding or removal of the inhibitors is reached when an increase in the efficiency is detected in the subsequent nucleic acid analysis reaction, wherein the efficiency may be before the inventive cleaning, especially in highly contaminated by inhibitors samples, at zero. However, the inhibitors can be assumed depending on the biological sample from the examination of the same biological sample due to the different amount and heterogeneity for comparison. For example, in stool samples, the material previously uncharacterized a particularly high content of having heterogeneous inhibitory substances, should have an efficiency of at least 40%, preferably at least 60% and especially at least 70% to be accessible in a quality test desirably. The efficiency is defined as the percentage of specific festzustellenden PCR products in a PCR analysis approach, in which a simple PCR reaction, but is advantageously carried out with the use of carrier protein, for example according to the examples below.

Another überrachendes result was due to a comparison with other eligible conventional basic anion exchanger materials, such as a FPLC Mono Q ™ system, the so-called GeneReleaser ™ system, a

Diethylaminoethyl cellulose ion exchanger (DE.AE-Sephacell ™; DE-52) or ™ Qiagen -Silika material: Although it is the same principle of anion exchange, the anion-exchange resin used in the invention binds the inhibitors from the biological starting material selective and effectively, so that the subsequent nucleic acid analysis reactions provide very high amplification rates already at .Application a simple PCR. According to the invention give much better results, namely already when using a smaller amount of anion exchange resin and, further, a lower number of extraction steps are required; usually an incubation sufficient. A comparison of conventional DNA purification and isolation was recently by A.Kramvis et al. described in "Journal of Clinical Microbiology", 34, 2731-2733 (1996).

The achievable with the invention to provide selective inhibitors of .Abtrennung is achieved even with unpurified output samples.

Thus, the inventive method has proven to be particularly effective in so far been very problematic starting samples, for example in whole blood was treated with citrate and EDTA or probably PCR-inhibiting substance heparin, as well as stool samples derived from the lysis of exfoliated in feces intestinal epithelial cells having a high and .Anteil to not known inhibitors. Of course, the advantages of the invention are achieved but also when applied to any other biological samples, such as other samples from the human body, such as body secretions, tissue samples, urine u. like.

The inventively used anion exchange resin is a basic, especially strongly basic polymeric material of high molecular weight (for example, in

Molecular weight range of from about 0.5 • 10 6, such as from 0.5 to

10 • 10 6, in particular 1 to 5 • 10 6) which is composed of a modified tertiary and / or quaternary ammonium groups organic polymer or copolymer, in general, and its characteristic feature is the above-described affinity for bile acids.

The resin-polymer is formed in a particularly suitable manner of a polystyrene obtained by divinylbenzene, preferably with a proportion of 0.5 to 5 wt .-%, particularly 1 - 3 wt .- crosslinked% divinylbenzene and in which network structure quaternary .Aitraioniumgruppen, are suitably incorporated in the form of benzyltrimethylammonium.

An example of an anion exchange resin these polymer type, which has a high affinity for bile acids and with the use of which excellent results were obtained, cholestyramine (average binding capacity of cholestyramine opposite cholic = 2.2 equivalent per kg resin dry weight) is. Cholestyramine is the international nonproprietary name for the plasma cholesterol level-lowering copolymers of styrene (vinylbenzene) and about 2% of divinylbenzene with inserted in the mesh structure quaternary .Ammoniumgruppen. The cholestyramine granules is known in the medical and therapeutic field as a highly hydrophilic, water-soluble, basic .Anionenaustauscherharz of binding bile acids in bile acid loss syndromes and as lipid-lowering agents for the treatment of hypercholesterolemia. The lipid-lowering drugs cholestyramine is sold by STADApharm.

Further examples of a, for use in the present invention basically good appropriate resin type form crosslinked polyalkylamine anion exchange resins. The crosslinked polyalkylamine .Anionenaustauscherharz is a poly usually as a copolymer (lower) alkyl polyamine, for example diethylenetriamine or Tertaethylenpentamin with epichlorohydrin (l-chloro-2, 3-epoxypropane) is formed.

An example of a .Anionenaustauscher resin these polymer type, which has a high affinity for bile acids and its use is also excellent results were obtained is colestipol (average binding capacity of Colestypol opposite cholic = 1.1 equivalent per kg resin dry weight) , Colestipol is the international nonproprietary name for the serum cholesterol lowering copolymers of diethylenetriamine and epichlorohydrin. The colestipol hydrochloride granules is also known per se as a water-insoluble, basic .Anionenaustauscherharz of binding bile acids in bile and hypercholesterolemia (lipid lowering). Colestipol was developed by Upjohn and came in 1977 to the market.

In correlation with the increased bile acid binding affinity purification efficiency compared to the inhibitors in the use of cholestyramine compared to colestipol is further increased.

The the resin constituent polymers include, in addition to the aforementioned copolymers, other polymers and copolymers and the use of substituted representatives of the copolymers constituting, exemplified monomers, provided in .Analogie with the binding capacity for bile acids that may be inhibitors present in the by enhancing the efficiency subsequent nucleic acid analysis reaction are bound lockable dimensions and separated.

Furthermore, the inventively used

Anion exchange resins further contain known additives in conventional amounts, such as flow agents. Ladungsabsättigung to the anion exchange resin also contains suitable salt partners; tertiary ammonium groups are suitably in the hydrochloride form, when quaternary ammonium groups are suitably associated with chloride.

Prior to the reaction of the sample to be used, the resin can be initially present in a suitable dry form, for example as granules, or may be already in an aqueous

present suspension. Is understood from the dry resin, it is recommended to pre-swell the resin in a suitable buffer, in order to avoid losses of the fluid volume of the biological sample used because of the hygroscopic properties of the resin. The resin suspension used for the reaction is usually buffered, suitably in the range from slightly acidic (about pH 5-6) to slightly alkaline (about pH 9-10).

As nucleic acid are all types of nucleic acid, particularly DNA and RNA in question. Because of the greater importance, but especially because nucleic acid amplification reactions are usually built on DNA samples (see., PCR), the invention will hereinafter be further described representative especially for cleaning or isolation of DNA.

In initial biological sample in which the nucleic acid to be isolated is present intracellularly, such as exfoliated epithelial cells stool samples containing the cells must first be lysed to unlock the intracellular material.

The lysis or disruption of the cells can be achieved by a simultaneous physical and chemical action on the body cells containing sample. The sample material may be present prior to lysis in a frozen state (-80 ° C). In the lysis buffer is suitably a denaturing agent, such as sodium dodecyl sulfate containing (SDS) or other detergents which causes the chemical disruption of the cells, while stirring the suspension favored, for example, with an automatic, mechanical stirring system, the mechanical pulping. In stool samples, it has been found that a vigorous mixing of the sample facilitates the case of very firm consistency with a test tube shaker for the downstream applications. By a centrifugation cell debris, undigested food residue or other macro-residues can be removed. .Anschließend lysis, treating the nucleic acid-containing cell lysate with the inventively used, special anion-exchange resin is carried out. The resin can be preswelled in this case suitably in the lysis buffer.

It has been found that already allowed for a unique reacting the anion exchange resin used in the invention, a sufficient cleaning of the selective inhibitors. The amount of the particular resin used is preferably 10 wt .-% or less, based on the total weight of the treated sample. Above this amount may tend, that not only the inhibitors, but also the desired nucleic acid is bound to an increasing extent to the resin.

It has been found that too frequent repetition of the batch-wise extraction, in particular with a high amount of use of about 10 wt .-% bile acid binding resins can lead to a very strong adverse reduction of nucleic acid quantity. The reason for this is unclear. It is believed that the particular resin initially binds the inhibitors and is saturated thereby. Missing, however, the inhibitors in the aqueous solution, the specific resin can help retain the nucleic acid due to its charge characteristics.

An aqueous to 10% strength by weight, particularly 5 to 10 weight-% suspension of the resin binds the inhibitors in sufficient quantity and at the same time sets the amount of the present DNA in the aqueous solution only slightly or not reduced.

Suitably, this is the amount of resin used to the frequency of transposition in an inverse relationship. That is, in a relatively high content, in particular 7.5 to 10 wt .-% resin suspension in an aqueous medium is preferable a one-time implementation, while in the medium content range, such as from 2.5 to 7.5 wt .-% and in particular to 5 wt .-% (± 1 wt), a two- or multi-time implementation provides better results. but preferably to be selected ratio of resin content to frequency of implementation also depends on the particular sample material to be examined. Thus, in stool samples a unique reaction with 10 wt .-% or two times of reacting with 5 wt .-% copolymer resin is suitable. In whole blood content ranges are less than 5 Ge preferable .-%, with a twice reacting each with 2.5 wt .-% copolymer resin is particularly suitable.

In reacting the nucleic acid-containing (may not be pre-cleaned) sample is most easily with the resin suspension was added, mixed very well and then separated again by Copolymeren- resin. The latter can be done easily by a .Abzentrifugieren the resin granules. but the reaction can also be effected by means of a separation on a the specific anion-exchange resin onset chromatography system.

Following the Inhibitorabreinigung the DNA can be further purified and isolated. It seemed appropriate to use proteinases initially unspecific acting as the proteinase K to degrade proteins and nucleic acid splitting enzymes is advantageous. Thereafter, one obtains a viscous, jelly-like liquid. From this, the DNA is preferably isolated by phenol extraction and subsequent ethanol precipitation from the aqueous phase. Alternatively, the DNA can be by the use of a detergent, chaotropic for example, a guanidine-containing detergent (such as DNAzol ™ from Gibco BRL) isolating followed by ethanol precipitation from the aqueous phase. For this purpose, the DNA-containing solution with proteinase K digested with the detergent and ethanol and immediately pelleted by a centrifugation step. Since neither organic solvents (phenol, chloroform) are used, still more Zentrifugationssschritte are needed for extraction, this method is very user-friendly and Second saving. The use of organic solvents such as phenol or chaotropic detergents for the isolation of DNA may alternatively be replaced by the use of known for this purpose centrifugation column, for example by QIAamp spin columns ™ from Qiagen ™. Here, both a Proteinase K digestion, as well as the use of commercially available lysis buffers (z. B. AVL buffer of QIAamp Viral RNA Kit from Qiagen ™, hepatitis C virus lysis reagent of the Amplicor HCV Kits ™ Hoffmann-La suitable for cell lysis Roche AG). When using the commercially available lysis buffer treatment of the samples similar to the respective protocol of the manufacturer occurs.

The purification or isolation of the DNA or RNA can then - as desired - subsequent reactions connect. The required quality of the nucleic acid sample is provided by the inventive methods. The procedure of the invention ensures preparation of the nucleic acid with a high yield (for example, 10-20 micrograms of DNA per 200 mg of stool sample) and purity (A o 26 / 28o 1/7 = - 1.9) under .Abreinigung of inhibitors of enzymatic reactions and allows to perform a high reproducible .Analytik, in particular in combination with enzymatic method for amplifying DNA.

It has been found that the inventive method can be combined in a particularly favorable manner with a PCR amplification using already carried out a simple PCR in more than 60%, partially more than 80% of all the stool samples to success.

In a .Amplifikation the isolated chair DNA by a nested PCR, the expected .Amplifikat could be obtained even in up to 100% of the tested samples.

The purified or in accordance with the inventive cleaning method isolated DNA PCR is preferably subjected, in the presence of a carrier protein such as bovine serum albumin (BSA), is carried out. The carrier protein concentration is high, preferably more than 50 ug / ml. Very good amplification rates have at carrier protein concentrations in the range of 120 - yield 200 ug / ml. Furthermore, relatively high concentrations required to act for the PCR nucleotides (deoxyribonucleoside

Triphosphate), primers and DNA Polymeräsen as the tag DNA polymerase advantageous. The nucleotide concentrations are preferably in the range of 150-225 uM. Simultaneously, the primer concentration is in the range from 0.75 to 1.25 uM. The content of DNA polymerase is suitably in the range of 2 to 3 units of 50 ul per approach.

The amplification is done with regard to the intended routine use in diagnostic laboratory preferably by a simple PCR, are run in the 30-35 temperature cycles.

The invention is further illustrated by the following examples. example 1

Isolating DNA from a stool sample:

200 mg of stool material is frozen for the duration of at least one hour at -80 ° C, then 600 ul of 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 × g in a tabletop Eppendorf centrifuge to separate coarse particles chair, cell debris, bacteria and food residues. The supernatant is a second time at 4 ° C, 20000 × g for 10 min. centrifuged. The DNA-containing supernatant is mixed with the same volume of a solution of cholestyramine (5% cholestyramine in lysis buffer), mixed well, 2 min. and incubated at room temperature as described above at 20,000 xg. After repeating this extraction step, the clear supernatant with Proteinase K is incubated (final concentration 100 ug / ml) for 2 hours at 56 ° C. The digested sample is mixed with an equal volume of phenol-chloroform-isoamyl alcohol solution (25: 1: 24) was added, well mixed, and 5 min. centrifuged at room temperature and 20000 xg. The aqueous upper phase is once more with an equal volume of a chloroform-Isoa ylalkohol solution: mixed and centrifuged as described above (24: 1). From the aqueous upper phase, the DNA can be pelleted by ethanol precipitation, by adding 1 / 10th volume of 3 M sodium acetate, pH 5.2 and 2.5 volumes of 100% ethanol. The fixed in 75% ethanol washed and dried at room temperature the DNA pellet is dissolved in 100 .mu.l of distilled water. The DNA yield is 10-15 micrograms per 200 mg of stool sample with an A 260/2 EO ratio of 1.7. 5 ul of this DNA solution are defined for the amplification of genes / gene segments used in a 50 ul PCR approach. The amplification mixture is composed as follows:

10 M Tris-HCl, pH 8, 3

50 mM KC1

2, 0 mM MgCl2

200 uM each dNTP

160 ug / ml bovine serum albumin (BSA)

1 uM each primer

2.5 U Taq DNA polymerase per 50 ul approach

example 2

Alternative isolation of DNA from a stool sample

200 mg of stool material is for a period of at least one hour at - 80 ° C frozen, followed by 600 .mu.l of 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 × g in a tabletop Eppendorf centrifuge to separate coarse particles chair, cell debris, bacteria and food residues. The supernatant is a second time at 4 ° C, 13000 × g for 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. incubated at room temperature and at 13,000 xg as described above. After repeating this extraction step, the clear supernatant with Proteinase K is incubated (final concentration 100 ug / ml) for 2 hours at 56 ° C. The digested sample is mixed with an equal volume of phenol-chloroform-isoamyl alcohol solution (25: 1: 24) was added, well mixed, and 5 min. centrifuged at room temperature and 13000 xg. The aqueous upper phase is once more with an equal volume of a chloroform-isoamyl alcohol solution (24: 1) was added and centrifuged as described above. From the aqueous upper phase, the DNA can be obtained by ethanol precipitation, by adding 1 / 10th volume of 3 M sodium acetate, pH 5.2 and 2.5 volumes

Of 100% ethanol are removed. The fixed in 75% ethanol washed and dried at room temperature the DNA pellet is dissolved in 100 .mu.l of distilled water. The DNA yield is 15-20 micrograms per 200 mg of stool sample with a A 260 / A 280 ratio of 1.9. 5 ul of this DNA solution are defined for the amplification of genes / gene segments used in a 50 ul PCR approach. The amplification mix is ​​identical to that of Example 1. Fig.

example 3

Isolation of DNA from whole blood:

500 ul of citrate, heparin or EDTA-blood, or frozen and then thawed blood are added (mM NaCl, 1% SDS, pH 9.0 500 mM Tris, 75 mM EDTA, 10) with 500 ul of lysis buffer and mixed well. The DNA-containing solution is mixed with the same volume of a solution of cholestyramine (2.5% cholestyramine in lysis buffer), mixed well, 2 min. at

incubated at room temperature and centrifuged 20,000 xg as described above. After repeating this extraction step, the clear supernatant with Proteinase K is incubated (final concentration 100 ug / ml) for 2 hours at 56 ° C. The digested sample is mixed with an equal volume of

added phenol, mixed well and 5 min. centrifuged at room temperature and 20000 xg. The aqueous upper phase is once more with an equal volume of a chloroform-isoamyl alcohol solution (24: 1) was added and centrifuged as described above. For efficient lysis of all eukaryotic and / or prokaryotic cells and / or viruses (simultaneous inactivation of infectious pathogens) and by denaturation and enzymatic degradation of proteins (simultaneous removal of the bound to the nucleic acid proteins), the DNA from the aqueous upper phase by ethanol precipitation are pelleted by adding 1 / 10th volume of 3 M sodium acetate, pH 5.2 and 2.5 volumes of 100% ethanol. The fixed in 75% ethanol washed and dried at room temperature DNA pellet is dissolved in 30 ul distilled water. The DNA yield is 5 - 10 ug per 500 ul of whole blood with a Ä 2 6o / 2 8o ratio of 1.7. 5 ul of this DNA solution are defined for the amplification of genes / gene segments used in a 50 μl- PCR approach. The amplification mix is ​​identical to that of Example 1. Fig.

Claims

claims
1. A method for cleaning, possibly also analysis of nucleic acids from biological samples, characterized in that the method comprises the step that the nucleic acid containing sample is reacted with an anion exchange resin which has a binding affinity for bile acids, which may inhibitors present are bound for the optional subsequent nucleic acid analysis reaction to the anion exchange resin and separated.
2. The method according .Anspruch 1, characterized in that the anion exchange resin comprises a polystyrene copolymer that has been crosslinked with divinylbenzene and made strongly basic by the incorporation of tertiary and / or quaternary ammonium groups.
3. The method according to claim 2, characterized in that is used as a copolymer cholestyramine.
4. The method according to claim 1, characterized in that the anion exchange resin comprises a polyalkylamine resin.
5. The method according to claim 4, characterized in that the copolymer was formed from Polyethylenpolyamin- and epichlorohydrin monomers.
6. The method according .Anspruch 5, characterized in that is used as a copolymer colestipol.
7. The method according to any one of the preceding claims, characterized in that 10 parts by weight does not exceed the implementation of the content of the anion exchange resin in a resin suspension%.
8. The method according to any one of the preceding claims, characterized in that the nucleic acid after the reaction with the .Anionenaustauscher resin is isolated by an isolation step.
9. The method of claim 8, characterized in that the isolation step by phenol extraction followed by
Alcohol precipitation occurs.
10. The method according to any one of the preceding .Ansprüche, characterized in that the purified nucleic acid is subjected to subsequent Anal a polymerase chain reaction.
11. The method according to claim 10, characterized in that the polymerase chain reaction is carried out in the presence of carrier protein.
12. The method according to claim 11, characterized in that the carrier protein concentration is more than 50 ug / ml.
13. The method of claim 11 or 12, characterized in that the polymerase chain reaction mixture containing 120-200 ug / ml of carrier protein, 150-225 uM of each deoxyribonucleoside triphosphate used and 0.75-1.25 uM of each primer used.
14. The use of cholestyramine for purifying and / or isolating and optionally subsequent .Analysieren of nucleic acids from biological samples.
15. Use of colestipol for purifying and / or isolating and optionally subsequent .Analysieren of nucleic acids from biological samples.
PCT/EP1997/005129 1996-09-19 1997-09-18 Method for purifying and eventually analyzing nucleic acids from biological test samples WO1998012351A1 (en)

Priority Applications (4)

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DE1996138362 DE19638362C1 (en) 1996-09-19 1996-09-19 Isolation of nucleic acids from sample
DE19638362.5 1996-09-19
DE1997131670 DE19731670C2 (en) 1997-07-23 1997-07-23 A method for cleaning and, where appropriate, analysis of nucleic acids from biological samples
DE19731670.0 1997-07-23

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AU4775497A AU4775497A (en) 1996-09-19 1997-09-18 Method for purifying and eventually analyzing nucleic acids from biological test samples

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EP0915171A2 (en) * 1997-11-04 1999-05-12 Becton Dickinson and Company Sample processing method using ion exchange resin
WO2000071698A2 (en) * 1999-04-30 2000-11-30 Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh Mixture for neutralizing enzyme inhibitors
WO2001010554A2 (en) * 1999-08-09 2001-02-15 Bilatec Ag Laboratory robot and method and reagent kit for isolating nucleic acids
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

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Cited By (12)

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EP0915171A2 (en) * 1997-11-04 1999-05-12 Becton Dickinson and Company Sample processing method using ion exchange resin
EP0915171A3 (en) * 1997-11-04 2000-03-22 Becton Dickinson and Company Sample processing method using ion exchange resin
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
WO2000071698A2 (en) * 1999-04-30 2000-11-30 Connex Gesellschaft Zur Optimierung Von Forschung Und Entwicklung Mbh Mixture for neutralizing enzyme inhibitors
WO2000071698A3 (en) * 1999-04-30 2001-02-15 Connex Ges Zur Optimierung Von Mixture for neutralizing enzyme inhibitors
WO2001010554A2 (en) * 1999-08-09 2001-02-15 Bilatec Ag Laboratory robot and method and reagent kit for isolating nucleic acids
WO2001010554A3 (en) * 1999-08-09 2001-08-23 Mario Berger Laboratory robot and method and reagent kit for isolating nucleic acids
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
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

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