Classifications

• • 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
• • 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

Definitions

• the present invention relates to a method for stabilizing, purifying and / or isolating nucleic acids from biological materials by removing impurities, e.g. substances harmful to nucleic acids and inhibiting enzymatic reactions.
• This method is particularly suitable for the analysis, detection or isolation of nucleic acids in stool samples.
• a reagent kit suitable for carrying out the method according to the invention is also disclosed.
• nucleic acids from biological materials that are contaminated with substances that damage nucleic acids during storage and enzymatic manipulation of the nucleic acids, e.g. inhibit by amplification. It is therefore important for the usability of the nucleic acids contained in the biological materials for further analyzes that these substances are only present in very low concentrations or are removed entirely from the sample.
• a method for isolating nucleic acids from stool is disclosed in WO 93/20235.
• this method gives only low yields of nucleic acids.
• DNA-damaging and / or PCR-inhibiting substances are not separated.
• the isolated DNA can therefore not be stored for a prolonged period of time and amplification to increase specific gene sections of this DNA to be analyzed does not provide reproducible results.
• a particularly serious disadvantage of the known method is that the PCR amplification does not result in intact DNA fragments with a uniform sequence which are necessary for further analysis. In order to obtain this, a complex cloning of the amplified gene sections is necessary.
• Yet another disadvantage of the prior art process is that the highly harmful solvents phenol and chloroform must be used.
• One object underlying the present invention was therefore to provide a method with which nucleic acids in biological materials are stabilized against degradation and in which substances which inhibit the enzymatic manipulation of nucleic acids can be separated off.
• a method is to be provided which enables reliable isolation of nucleic acids from fecal samples.
• This object is achieved by a process for the purification, stabilization and / or isolation of nucleic acids from biological materials, which is characterized in that an adsorption matrix for binding impurities is added to a sample of biological materials containing nucleic acids, and then the nucleic acids are added if separated from bound impurities.
• the sample containing nucleic acids can be brought into contact with the adsorption matrix directly or after the sample has been taken up in liquid.
• the usability of nucleic acids isolated from biological materials, in particular DNA can be significantly improved by the method according to the invention.
• the addition of the adsorption matrix largely eliminates both substances which damage nucleic acids and substances which inhibit enzymatic manipulation.
• the nucleic acids stabilized by the method according to the invention can therefore be stored for a long time.
• reproducible results are obtained when the nucleic acids treated by adding an adsorption matrix are amplified, for example by PCR. This reproducibility is essential for the meaningfulness of the results obtained in the nucleic acid analysis.
• the high quality of the nucleic acids purified by the method according to the invention can be seen, for example, in the fact that they can be examined directly by sequencing or heteroduplex analysis. Cloning is not necessary. In addition, no harmful solvents have to be used in the method according to the invention.
• the adsorption matrix used in the method according to the invention is designed in such a way that it contaminates nucleic acids and / or prevents the implementation of enzymatic reactions and / or inhibits enzymatic reactions, such as degradation products of hemoglobin, e.g. Bilirubin and its degradation products, or / and bile acids or salts thereof or their degradation products and other degradation products of plant or animal origin, can bind.
• An insoluble adsorption matrix is preferably used, since in this case an easier separation from the sample is possible.
• an adsorption matrix based on carbohydrates and / or polypeptides Good results are obtained with an adsorption matrix based on carbohydrates and / or polypeptides.
• An adsorption matrix based on carbohydrates for example an adsorption matrix containing polysaccharides, is preferred. It is particularly preferred to use an adsorption matrix which contains tx- or / and / S-glycosidically linked carbohydrates, for example starch, Cellulose, glycogen and / or other biogenic or nonbiogenic carbohydrates and derivatives or mixtures thereof.
• flours i.e. essentially a mixture of cellulose, starch, lipids and salts or components thereof.
• Flours made from cereals, corn, peas, soybeans and potatoes or components thereof or mixtures thereof have proven to be suitable, for example.
• other types of flour or mixtures of several types of flour or components thereof can also be used.
• Most preferred is the use of potato flour or components thereof. Mixtures of purified carbohydrates, e.g. Cellulose and flours e.g. Potato flour.
• an adsorption matrix based on carbohydrates together with soluble constituents made from flours, in particular from one or more of the above-mentioned types of flour.
• the amount in which the adsorption matrix is added to the biological sample depends essentially on the nature of the sample, i.e. the amount of contaminants. Good results have been obtained if the adsorption matrix is used in a weight fraction of 0.05: 1 to 100: 1 with respect to the sample containing nucleic acids.
• the adsorption matrix is particularly preferably added in an amount of 0.1: 1 to 10: 1.
• the sample containing nucleic acids comes from biological materials which contain impurities which degrade nucleic acids or inhibit enzymatic reactions.
• the sample containing nucleic acids preferably comes from faeces. However, it can also be obtained, for example, from other sources, for example tissues of any kind, bone marrow, human and animal body Liquids such as blood, serum, plasma, urine, sperm, cerebrospinal fluid, sputum and smears, plants, plant parts and extracts, for example juices, fungi, microorganisms such as bacteria, fossil or mummified samples, soil samples, sewage sludge, waste water and Food. For example, degradation products of hemoglobin, such as bilirubin and its degradation products, or / and bile acids or salts thereof or their degradation products, but also other types of contaminants can be present as impurities.
• the sample can be incubated with the adsorption matrix at room temperature. The incubation period can be varied over a wide range.
• the adsorption matrix can e.g. can be separated from the sample by centrifugation.
• the adsorption matrix can be added directly to the sample, e.g. in the case of liquid biological samples.
• the sample can be passed over an adsorption matrix by centrifugation, by applying a vacuum or / and by means of gravity, the adsorption matrix then preferably being packed in a column.
• a particularly preferred aspect of the present invention is the analysis, the detection or the isolation of nucleic acids, in particular DNA, from stool samples.
• the method according to the invention provides clean and amplifiable nucleic acids from fecal samples which can be used to detect mutations, in particular tumor-specific DNA mutations.
• the method according to the invention is of great importance for tumor diagnosis, since it enables the specific detection of nuclear eukaryotic nucleic acids in the presence of impurities and large amounts of bacterial nucleic acids.
• tumors of the digestive tract in particular pancreatic or intestinal tumors, can be diagnosed earlier and more precisely. This diagnosis is made, for example, by examining oncogenes and / or tumor suppressor genes for tumor-specific DNA mutations. Since cells from tumors of the digestive tract are continually erased into the stool, it is possible to detect tumor-specific DNA mutations in the stool using the method according to the invention.
• the method according to the invention also allows the monitoring of therapies which have been taken in order to eliminate a tumor, and the regular and reliable implementation of tumor prevention examinations.
• the test for occult blood in stool does not, or only very rarely, produces false-positive results.
• the detection of mutations in genes that mutate in the adenoma stage, ie in a very early stage of tumor progression enables a much earlier and more specific diagnosis than the stool blood test.
• the tumor suppressor gene APC adenomatous polyposis Coli
• the ras oncogene can be used.
• Mutation analyzes of these two genes in DNA from stool samples can be used in particular to detect intestinal tumors, for example colon tumors, and pancreatic tumors.
• intestinal tumors for example colon tumors, and pancreatic tumors.
• other tumor-relevant genes can of course also serve as analysis objects for cancer diagnosis.
• non-translated repetitive sections of the genome can also serve as analysis objects in cancer diagnosis. These so-called microsatellite sections are amplified and the band pattern obtained by gel electrophoresis is compared with the band pattern of DNA from healthy body material of the same patient. Different band patterns can indicate the presence of a tumor.
• Another example of the use of the method according to the invention is an exact identification of persons by examining the nucleic acids purified from faeces or body material in the forensic analysis. For this purpose, repetitive polymorphic sections of the genome are amplified and the amplification products are separated by gel electrophoresis. The person in question can then be identified by comparing the band patterns obtained with the patterns of DNA from other suspicious or closely related persons.
• Another important application for the isolation of DNA from fecal samples according to the invention are zoobiological population genetic, evolutionary genetic and botanical studies and investigations of animals and plants. So far, such studies have very often failed due to the rarity of an animal species and the low probability of encountering the animals in question at a specific location. If the approximate location is known, an analysis of faeces left behind by the method according to the invention can provide important information on the degree of kinship of the animals provide covered hiking trails or on the food habits of the animals. Likewise, important diagnostic information about infections, for example bacterial or viral, can be derived from the analysis of faecal nucleic acids, for example by detecting microbial or viral nucleic acids.
• Another object of the present invention is a reagent kit for stabilizing and purifying nucleic acids from biological materials, comprising:
• the adsorption matrix can be in a packaged portioned form, e.g. filled in a column such as a centrifuge mini column.
• the reagent kit preferably contains additional agents for the purification of nucleic acids, which include, for example, mineral and / or organic carriers as well as optionally solutions, auxiliaries or / and accessories.
• Mineral components of carriers can be, for example, porous or non-porous metal oxides or mixed metal oxides, for example aluminum oxide, titanium dioxide or zirconium dioxide, silica gels, materials based on glasses, for example modified or non-modified glass particles or glass powder, quartz, zeolites or mixtures of one or several of the substances mentioned above.
• the carrier can also contain organic constituents which, for example, consist of latex particles which may have been modified with functional groups, synthetic polymers, such as polyethylene, polypropylene, polyvinylidene fluoride, in particular ultra-high molecular weight polyethylene or HD polyethylene, or mixtures be selected from one or more of the aforementioned substances.
• synthetic polymers such as polyethylene, polypropylene, polyvinylidene fluoride, in particular ultra-high molecular weight polyethylene or HD polyethylene, or mixtures be selected from one or more of the aforementioned substances.
• the carrier can, for example, be in the form of particles with an average size of 0.1 ⁇ m to 1000 ⁇ m. If a porous carrier is used, an average pore size of 2 ⁇ m to 1000 ⁇ m is preferred.
• the carrier can be, for example, in the form of loose beds of particles, filter layers, e.g. made of glass, quartz or ceramic, membranes, e.g. Membranes in which a silica gel is arranged, fibers or fabrics made of mineral carriers, such as quartz or glass wool, and in the form of latices or frit materials made of synthetic polymers.
• the reagent kit can also contain suitable solutions, e.g. Contain washing solutions or buffer solutions for taking up the sample.
• a buffer suitable for taking up a sample containing nucleic acids is, for example, a buffer system based on Tris-HCl pH 8.5-9.5, EDTA and optionally NaCl.
• a particularly preferred buffer, in particular for taking stool samples, contains 500 mM Tris-HCl pH 9, 50 mM EDTA and 10 mM NaCl.
• the reagent kit according to the invention can also contain auxiliary substances such as enzymes and other agents for manipulating nucleic acids, e.g. at least one amplification primer and enzymes suitable for the amplification of nucleic acids, e.g. a nucleic acid polymerase and / or at least one restriction endonuclease.
• auxiliary substances such as enzymes and other agents for manipulating nucleic acids, e.g. at least one amplification primer and enzymes suitable for the amplification of nucleic acids, e.g. a nucleic acid polymerase and / or at least one restriction endonuclease.
• the primers for the amplification of nucleic acids expediently come from the genes to be analyzed, ie for example from oncogenes, tumor suppressor genes and / or microsatellite segments. Enzymes and restriction endonucleases suitable for amplifying nucleic acids are known and commercially available. The present invention is further to be illustrated by the following example.
• adsorption matrices were tested: Immobilized bovine serum albumin (RSA), cellulose and potato starch (all from Sigma, Kunststoff, DE) and potato flour (honey, Postbus 45, 1540 AA Koog a / d Zaan, NL), in which it it is essentially an insoluble mixture of cellulose, starch, lipids and salts.
• RSA Immobilized bovine serum albumin
• potato starch all from Sigma, Kunststoff, DE
• potato flour honey, Postbus 45, 1540 AA Koog a / d Zaan, NL
• the DNA was then eluted from the spin column in a final volume of 150 ul distilled water and used until
• the yield of chromosomal DNA was determined by measuring the absorbance at 260 nm.
• a DNA loss due to degradation was measured after storage for 1 week at ⁇ 20 ° C. by analytical agarose gel electrophoresis and spectrophotometric analysis.
• b PCR was performed on DNA from ten different stool samples. The number of samples that could be analyzed by PCR is indicated.
• Buffer A 5.6 M guanidinium / HCl; 20% tween 20
• Buffer B 10 mM Tris / HCl pH 7.5; 100 mM NaCl; 70% ethanol
• Buffer C 10 mM Tris / HCl pH 9.0; 0.5 mM EDTA
• the bound nucleic acids were purified by washing twice with 2.5 ml of buffer B and eluted from the Qia-AMP midi column with 0.5 ml of buffer C and stored at -20 ° C. for further use.

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Cited By (17)

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Citations (5)

• 1995
  • 1995-08-16 DE DE19530132A patent/DE19530132C2/de not_active Expired - Lifetime
• 1996
  • 1996-08-14 AT AT96928466T patent/ATE215611T1/de active
  • 1996-08-14 DE DE59609027T patent/DE59609027D1/de not_active Expired - Lifetime
  • 1996-08-14 EP EP96928466A patent/EP0851937B1/de not_active Expired - Lifetime
  • 1996-08-14 WO PCT/EP1996/003595 patent/WO1997007239A1/de not_active Ceased
  • 1996-08-14 CA CA2228769A patent/CA2228769C/en not_active Expired - Lifetime
  • 1996-08-14 JP JP50894597A patent/JP4264133B2/ja not_active Expired - Lifetime
  • 1996-08-14 US US09/011,567 patent/US6084091A/en not_active Expired - Lifetime
  • 1996-08-14 DK DK96928466T patent/DK0851937T3/da active
  • 1996-08-14 AU AU68216/96A patent/AU712331B2/en not_active Expired

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