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WO1989000206A1 - Procede d'analyse de fluides biologiques pour depister de l'adn cellulaire oncogene ou des fragments de celui-ci - Google Patents

Procede d'analyse de fluides biologiques pour depister de l'adn cellulaire oncogene ou des fragments de celui-ci Download PDF

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WO1989000206A1
WO1989000206A1 PCT/DE1988/000384 DE8800384W WO8900206A1 WO 1989000206 A1 WO1989000206 A1 WO 1989000206A1 DE 8800384 W DE8800384 W DE 8800384W WO 8900206 A1 WO8900206 A1 WO 8900206A1
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dna
oncogene
labeled
product
plasma
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Viktor Balazs
Margit BALAZS-FRÖHLICH
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a method for the detection of cellular Onko ⁇ gene DNA, or fragments thereof, according to the preamble of claim 1.
  • the increased activity of two or more cellular oncogenes through mutation, amplification (amplification) or other disorders of gene regulation is a major cause of the malignant transformation and the diversification of the malignant cells.
  • Amplification of the cellular oncogenes on e.g. B. twice to a hundredfold may be the cause of the malignant transformation in question or occur during the progression or diversification of the malignant cells in cases of 30% to 50% of the malignant diseases. Not all types of oncogenes have a tendency to amplify. Interestingly, those oncogenes who tend to occur most frequently in malignant diseases in activated form have this tendency; three out of four in group A, two out of eight in group B and zero out of six in group C, the groups are given on page 16. In 15% to 30% of cases of malignant diseases, the oncogenes which arise from point mutation and are not present in the normal state can be detected in 3T3 NIH cells using the transfection method. It is recently known that the mutation plays an important role in a much higher percentage (40%) of the malignant cases.
  • a very significant disadvantage of this like any other method, which is based on the immunological detection of the proteins or polypeptides of cancer cell origin, is that the presence of specific antibodies, which are produced by the host or introduced for therapeutic purposes, cause unrealistic results and in general can be a significant disruptive factor.
  • the invention has for its object to provide a method for the detection of the DNA, or its fragments, from cellular oncogenes in a cellular biological fluid, such as blood plasma. Since it is necessary for this to first separate the DNA from the blood plasma, it is a further object of the invention to provide a reliable method for separating the DNA from the blood plasma.
  • the DNA that has entered the bloodstream and whose fragments can be extracted from the blood plasma, its oncogene content can be detected by in vitro molecular hybridization.
  • the detection of oncogenes or their fragments in blood plasma by in vitro hybridization is all the more suitable for this purpose because they are not only extremely specific but also highly sensitive and as little as 0.5 to 2 pg DNA / ml are specifically detected can.
  • the combination of the in vitro amplification of the target sequences of the oncogenes with the in vitro hybridization increases the sensitivity of the method by more than two orders of magnitude.
  • the total plasma DNA like the DNA that has entered the bloodstream, or fragments thereof, are isolated from the blood plasma of patients with malignant and non-malignant diseases and examined for the presence of the DNA of cellular oncogenes with in vitro molecular hybridization.
  • the qualitative and quantitative differences enable the method according to the invention for examining the oncogene DNA in the blood plasma to be used as evidence of active malignant processes.
  • the practical applicability of the method for the determination of the DNA, or its fragments, of cellular cancerogens from human blood plasma is a universal malignancy test for the detection of malignant Processes.
  • the most important advantage of this invention is that it specifies a method that can serve as a universal malignancy test: This test is based on the basic principles of the malignant transformation by the activation of cellular oncogenes and the special features of the malignant cells and tumors.
  • the second significant advantage of this method is the high sensitivity (1 pg / ml).
  • Another major advantage of the invention in contrast to irrigation tests, is that the specific antibodies which have been formed by the host or which have been introduced into the bloodstream for therapeutic purposes cannot influence the results.
  • the method according to the invention has the advantage that there is no interference with the result by specific antibodies.
  • This method also has the advantages that the risk of DNA degradation is significantly lower.
  • the greatest advantage is that the detection of the oncogene DNA is able to prove that the activation of the oncogene was caused by point mutation and which point of the oncogene was changed by mutation. For this reason, you can know how the oncogene product (OKP) is changed. All ras oncogenes can be changed in position 12, 13, 61 by point mutation. The mutation particularly affects
  • This change leads to an oncogene product in which the amino acid gly in position 21 (p21) with val (especially in colon and bladder cancer), arg (especially in Lung cancer) or confused with cys (especially in lung cancer).
  • Oligonucleotide samples for these oncogenes for the purpose of this hybridization are commercially available (Oncogene Science Inc., Mineola, NY 11501).
  • this oncogene product is only present in the tumor cells and is not present at all in the normal cells, this mutated oncogene product offers a unique, exclusive diagnostic point and therapeutic point of attack on the malignant cells.
  • the basic character of malignant processes and their behavior in the host is largely determined by which oncogenes have been activated (genetic scripture, cell oncogene profile). Until now, this could only be determined histologically in tumor tissues by in vitro nucleic acid hybridization.
  • the malignancy test according to the invention can provide this information at least partially from the blood plasma (plasma oncogene profile) long before the relatively small tumor cell mass can be visualized and reached for histological examinations.
  • the detection of the oncogene DNA is particularly suitable for examining and observing the amplification of the oncogenes.
  • an intensification (amplification) of the activated oncogenes can occur, which can cause a significantly increased aggressiveness and progression of the malignant cells.
  • the occurrence of such an enhancement can be observed early on during the follow-up with the quantitative maglignity test.
  • This means that a subclone of the tumor cells, which has the amplified oncogene, can be recognized at an early stage before it has been able to spread and could worsen the prognosis.
  • This subclone can be influenced in the early stages by specific immunotherapy or ___ m_ ⁇ onochernot_herapie and possibly destroyed.
  • Amplification of the cellular oncogenes occurs either in the primer or during the progression and diversification of the malignant cells and in approximately 30% to 50% of the cases of malignant diseases.
  • Gene products of the activated cellular oncogenes play an important, even vital function in the transformed malignant cells, many of them act as protein kinase enzymes in the cell membrane.
  • the change or impairment of this function by attacking the malignant cells in an immunospecific manner, targeting their oncogene products with monoclonal antibodies alone or in conjunction with substances having a radioactive or chemotherapeutic effect, can lead to the curative effect before the tumor cell mass could spread.
  • a plasma oncogene profile with early detection shows the specific therapy options.
  • follow-up with new oncogene samples enables new oncogene DNA to be recognized.
  • a quantitative malignancy test during the course of the disease offers an early detection of a possible amplification of a cellular oncogene and gives specific information on how to suppress or destroy this new subclone of malignant cells before a larger progression or aggressiveness is caused.
  • the plasma is quickly separated from the freshly obtained blood. Then the plasma is mixed with a detergent such as sodium duodezyl sulphate (SDS) and a proteolytic enzyme such as Proteinase K (Boehringer Mann ⁇ heim) with an aqueous medium and is then at 37 ° C for 1 to 3 h in a buffer solution (pH 7 , 5) incubated. The viscous mixture is periodically stirred by vortexing. After the incubation, extraction is carried out three times using organic solvent, such as phenol. The result is an organic phase that accounts for the largest share of the proteins, and an aqueous phase that contains essentially all of the DNA. Phenol is removed after centrifugation.
  • a detergent such as sodium duodezyl sulphate (SDS) and a proteolytic enzyme such as Proteinase K (Boehringer Mann ⁇ heim)
  • SDS sodium duodezyl sulphate
  • proteolytic enzyme such as Proteinase K (Boehringer Mann
  • the interphase is re-extracted with additional phenol and buffer.
  • the DNA is incubated in the presence of 0.25 M Na acetate at pH 5.2 with ice-cold ethanol at -20 degrees C, precipitated and centrifuged.
  • the DNA sediment (which is not always visible) is dissolved in the desired buffer or solution.
  • the DNA is subjected to DNase enzyme-free RNase treatment at 37 degrees C for 1 h.
  • the DNA is then extracted twice with phenol / chloroform and precipitated with ethanol as above and dissolved in the desired solvent.
  • the specific target sequences of all oncogenes can be significantly increased specifically by enzymatic amplification in vitro.
  • the plasma DNA is subjected to such an in vitro enzymatic amplification by means of polymerase chain reaction (PKR) in order to significantly increase the specific sequences changed by point mutation.
  • PSR polymerase chain reaction
  • Two oligonucleotide DNA fragments (each of 20 bases), complementary to the sequences flanking the target codon, are used as primers to the adjacent sequences, which have the mutated codon, by the Klen ⁇ w fragment of Escherichia coli DNA- Copy polymerase I according to the principle of Saiki and co-workers (Science 230: 1350 - 1354, 1985). Annealing of the oligomer primers to the corresponding denatured oncogene DNA from the blood plasma is continued by synthesis of the new fragments which contain the target sequence under the action of the enzyme and in the presence of deoxynucleotide triphosphate.
  • the newly synthesized target sequence serves as another template strand for the oligonucleotide primers and for the enzyme. Repeated cycles of denaturation, primer annealing and chain elongation cause an exponential increase in the target sequences. So z. B. 20-25 cycles amplification in vitro in 2 to 2.5 an approximately 200,000-fold increase in the specific target sequences.
  • the DNA is hybridized in vitro with labeled oligonucleotide oncogene samples which have the mutated codon.
  • the hybrid duplexes formed in this way are stable when the match is perfect. In the case of mismatch, the hybrid duplexes are not as stable.
  • the DNA dissolved in the water is denatured by heating to 100 ° C. for 10 min and by mixing with the same volume of 1 M NaOH and at room temperature Incubated for 20 min.
  • the alkali is then neutralized to a neutral value (pH 8.0) by admixing 1 M HCl and the salt concentration is reduced by adding 1 M NaCl, 0.3 M Na citrate, 0.5 M Tris.CL (pH 8 , 0) increased. Then it is cooled in ice.
  • the increase in the salt concentration serves to increase the binding capacity of the DNA to the solid substrate (see below).
  • the resulting solution of denatured DNA is applied in the desired amount on a solid substrate, such as pure nitrocellulose. gene to cause immobilization of the denatured DNA on the solid substrate.
  • the resulting solid substrate is washed at room temperature with 50 ml vol 6 x SSC and dried, then baked in a vacuum oven at 80 degrees C for 2 h. The baked solid substrate is then treated to prevent further binding of nucleic acid to the solid substrate.
  • the (radioisotopically or non-radioisotopically) labeled oncogene DNA sample is denatured and brought into contact in a solution with the solid substrate on which the plasma DNA is already immobilized in order to hybridize it under selected thermal and chemical conditions. This causes the labeled oncogene DNA sample to be (hybridized) by hydrogen bonds with the complementary sequence (s) of the plasma DNA immobilized on the solid substrate, if they have these co-complementary sequences.
  • the solid substrate is subjected to washing in order to remove the non-specifically bound, labeled oncogene DNA sample.
  • the solid substrate is then subjected to an analysis in order to detect the occurrence of the hybridization between the plasma DNA and the marked oncogene DNA sample.
  • an analysis in order to detect the occurrence of the hybridization between the plasma DNA and the marked oncogene DNA sample.
  • radioisotopic labeling this is determined by autoradiography, in the case of non-radioisotopic labeling by detection of the labeling substance by means of an enzyme affinity test by color reaction.
  • Radioisotopes that can be used for this procedure are, for example:
  • P 125 131 3 especially 32 but also I, I and H.
  • the non-radioisotopic labeling of the oncogene DNA samples is carried out, for example, with biotin, also by nick translation (Proc. Natl. Acad. Sci. USA 78: 6633-6637, 1981) and most recently with photobiotin (Nueleic Acid Research 13: 745- 761, 1985). Labeling with photobiotin is particularly straightforward, quick and inexpensive. A photobiotin labeling and detection kit is commercially available (BRESA, Sydney, South Australia). Labeling with biotin has a very important advantage: apart from the fact that it is not radioactive, results can be seen within a few hours by color reaction. You don't have to wait days, like with autoradiography.
  • Oncogene DNA samples oncogene oligonucleotide samples, labeled or unlabeled, are commercially available (ONCOR, Inc. Gaithersburgh, Ma. USA 20877; ONCOGENE SCIENCE Inc. Mineola, NY. 11501 USA).
  • Tris.Cl pH 7.5
  • EDTA ethylenediaminotetraacetic acid
  • NaCl 0.6 M
  • SDS sodium duodecyl sulfate
  • proteinase K final concentration 200 micrograms / ml, Boehringer
  • sodium acetate of 2.5 M (pH 5.2) is added to the aqueous phase in order to reach the final concentration of 0.25 M.
  • Two volumes of ice-cold ethanol are added, mixed well and incubated at -20 degrees C and then centrifuged.
  • the DNA sediment (not always visible!) Is dissolved in TE (pH 8.0) (10 M Tris.Cl pH 8.0; 1 M EDTA pH 8.0) and with DNase-free RNase (final concentration 10 micrograms / ml) incubated at room temperature for 60 min, then extracted twice with phenol-chloroform and precipitated with ethanol as above.
  • TE pH 8.0
  • DNase-free RNase final concentration 10 micrograms / ml
  • the plasma DNA ' dissolved in water in the desired concentration, is heated to 100 ° C. for 10 min, then quickly cooled in ice, mixed with the same volume of 1 M NaOH and incubated at incineration temperature for 20 min . The denaturation of the DNA was thus achieved.
  • the denatured DNA is mixed with a solution of 1 M NaCl, 0.3 M Na citrate, 0.5 M Tris.Cl (pH 8.0) and 1M HCl, then cooled in ice.
  • a corresponding volume of the resulting solution (10 micrograms of DNA) is applied to a solid substrate such as pure nitrocellulose, the corresponding volume of the denatured DNA solution being introduced into a well of a "microfilter” plate with 96 wells (minifold I Filtration and Incubation Plate, Schleicher & Schuell, Keene, New Hampshire, 03431, USA) (Bio-Rad, Richmond, Ca, 94 804, USA) and filtered under a gentle vacuum to form a 3.0 mm diameter spot becomes.
  • the resulting filter is washed with a solution of 50 ml of 6 x SSC at Zir ⁇ merte ⁇ peratur, dried and baked at 80 degrees C for 2 h in a vacuum oven.
  • Prehybridization solution For amide 50% (Völ / Vol); 5 x Denhardt's solution; 5 x SSPE; 0.1% SDS; 100 micrograms / ml denatured DNA from herring sperm and 1 microgram / ml poly A. (Denhart's solution: Ficoll 5 g; polyvinyl pirrololidone 5 g; bovine serum albumin (Pentax fraction V. 5 g and H ⁇ 0 to 500 ml.) The filter is incubated in this prehybridization solution at 42 ° C. for 6-8 h. (20 ⁇ SSPE: 174 g NaCl; 27.6 g NaH 2 PO 4 ; 7.4 g EDTA ad 1 liter HO, pH 7.4).
  • the labeled DNA oncogene sample is denatured by heating to 100 degrees C for 5 min and quickly cooled in ice. In denatured form, the pre-hybridization solution in which the filter is already incubated becomes is mixed and further incubated at 42 degrees C for 12-20 h.
  • the solution in which the incubation was carried out is poured away and the filter is subjected to washing 3-4 times for 5-10 minutes (per wash) at a cirrhotic temperature in a large volume of 2 ⁇ SSC and 0.1% SDS .
  • the filter is subjected to two more washes for 1 - 1.5 h in a solution of 1 x SSC and 0.1% SDS at 68 degrees C. If the background is still high, further washes with higher stringencies (sharpening) are necessary: for 60 min in a solution of 0.2 x SSC and 0.1% SDS at 60 degrees C.
  • the samples were radioisotopically labeled with P 32 (specific activity: 10 cpm / mi rograrrm) by nick translation according to Rigby et al (J. Mol. Biol. 133: 237-251, 1977) or obtained unlabeled.
  • the unlabeled DNA samples were either labeled with biotin (according to Leary, Proc. Nat. Acad. Sci. 80: 4045-4049, 1983) or with photobiotin.
  • Reagents for non-radioisotopic labeling by nick translation according to Rigby et al J. Mol. Biol.
  • the nitrocellulose paper is inserted between clear acetate foils and brought close to a film that is sensitive to the X-rays.
  • An intensifying screen is attached on the opposite side and packed light-tight. After a certain time, the film is developed. The presence of the DNA or its fragments that hybridized with the oncogene DNA sample is determined by the presence of a spot on the film.
  • the DNA samples are applied in 0.1 M EDTA, otherwise the prehybridization is carried out as with radioisotopically labeled DNA samples, but the duration of the Prehybridization is shorter: 4-8 h.
  • the hybridization is carried out as with radioactive samples, but at a higher temperature (55 degrees C) for 20 h in a solution: 4 vol. Prehybridization buffer; 1 volume of about 0.5 g / ml sodium dextran sulfate and 20 ng / ml biotin-labeled DNA sample (oncogene DNA sample).
  • the dried nitrocellulose papers are at 42 degrees C for 30 min in SIMT buffer (1 M NaCl; 0.1 M Tris. Cl. PH 7.5; 2 mM MgCl- 0.05% v / v Triton X-100) with 30 mg of Bovin serum albumin incubated. After the nitrocellose paper has dried, it is incubated at room temperature for 10 min in SIMT buffer with 1 microg / ml Sigma avidin-alkaline phosphate complex, then it is shaken frequently in SIMT buffer (3 x 10 min) and STM buffer (1 M NaCl, Tris. Cl pH 9.5, 5 mM MgCl) for 2 5 min incubated.
  • the nitrocellulose paper at room temperature in the dark with substrate solution (STM buffer but only with 0.1 M NaCl) with 0.33 mg / ml nitro blue tetrazolium, 0.17 mg / ml 5-bromo-4-chloro-3-indolyl Phosphate and 0.33% v / v N, N-dimethylformamide mixed.
  • substrate solution STM buffer but only with 0.1 M NaCl
  • 0.33 mg / ml nitro blue tetrazolium 0.17 mg / ml 5-bromo-4-chloro-3-indolyl Phosphate and 0.33% v / v N, N-dimethylformamide mixed.
  • the reaction is carried out by washing the nitrocellulose paper with 10 mM Tris.Cl pH 7.5; 1mM EDTA terminated.
  • 3-5 micrographic plasma DNA is composed to 300-500 microliter buffer from 10 mM Tris.HCl, pH 7.5, 50 mM NaCl, 10 irM MgCl, 1.5 mM deoxynucleotide triphosphate (dBTP, all four), 1 UM each of the two primers mixed in a plastic centrifuge tube.
  • the plasma DNA is denatured by heating at 95 degrees C for 5 min, then centrifuged to remove the condensation.
  • the tubes are incubated immediately at 30 degrees C for 2 min in order to be able to bind the two oligonucleotide primers to their target sequences by hybridization (annealing).
  • the dried filters are evaluated by autoradiography at -70 degrees C with a reinforcement label (for the night).
  • the DNA in denatured form is freely mixed in solution with the labeled oncogene DNA sample, incubated for 1-2 hours at 70 ° C. in the presence of nuclease inhibitors. Then hydroxapatite is added, incubated for 5 min at 70 degrees C in order to adsorb the product of the hybridization (plasma-DNA-labeled oncogene-DNA hybrid complexes).
  • the resulting hydroxyapatite fraction is separated as sediment by centrifugation.
  • the sediment is washed for 5 min at 70 degrees C and the product compared to the background in the hydroxyapatite fraction determined based on the label.
  • the procedure according to the invention for the detection of the DNA or its fragments, of cellular oncogenes in human blood plasma comprises several favorable reaction methods and steps in order to result in a high reliability and reproducibility.
  • Proteinase K facilitates the removal of the proteins, the guanidinium isothiocyanate treatment, the denaturation of the proteins and the cleavage of the DNA-protein complexes.
  • the rest of the proteins are removed by organic extraction.
  • the degradation and removal of the RNA is achieved by RNase treatment, because otherwise the RNA could interfere with the hybridization.
  • In vitro amplification enables a substantial increase in the target sequence and thus increases the sensitivity of the method by two orders of magnitude.
  • Baking ensures the reliability of the tight binding of the DNA to the filter. Treatment of the filter after baking prevents unspecific binding and also ensures reliability. Washing the filter after hybridization removes unnecessary, or non-specifically bound, labeled oncogene DNA samples and also contributes to reliability.
  • a semi-quantitative evaluation can also be carried out.
  • the intensity of the black spot on the X-ray film is measured by densitometry and quantitative comparisons can be made (reflectance densitometer, Bio-Rad, Richmond CA 94804, USA).
  • a blood plasma DNA with an oncogene sample has shown a positive test
  • the relative amount can be stormed by the dilution method.
  • a 1: 2 serial dilution is made from the blood plasma DNA and each dilution is tested by hybridization.
  • the highest dilution from which a positive signal is still emitted is the titer.
  • quantitative comparisons can be carried out during the observation period. In this way you can e.g. B. observe the amplification of one of the activated oncogenes: If the titer of an oncogene is increased disproportionately compared to other oncogenes during the follow-up, this means the amplification of this oncogene.
  • a positive test in the case of a malignant disease is usually obtained when the plasma DNA hybridizes only with oncogene group A (see above), because these oncogenes are activated in all or almost all types of malignancy. It is less common to extend hybridization to Group B.
  • oncogenes are particularly often activated in this cancer. New oncogenes are still being discovered. In the future, this may also be used as a sample.

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Abstract

Selon le procédé décrit, on concentre ou sépare l'ADN contenu dans un fluide biologique acellulaire complet, de préférence du plasma sanguin, on dénature le produit d'ADN ainsi obtenu et on l'immobilise sous cette forme sur un support solide ou on le laisse libre dans une solution. On met l'ADN en contact avec de l'ADN oncogène marqué ou avec des sondes à oligonucléotides, afin d'hybrider ceux-ci avec l'ADN lorsque des séquences complémentaires sont présentes. On elimine l'excédent d'ADN oncogène marqué ou de sondes à nucléotides n'ayant pas formé des liens spécifiques et on analyse le produit pour déterminer la présence d'ADN ou d'oligonucléotides marqués. On peut procéder à une amplification enzymatique in vitro avec l'ADN du plasma, après l'avoir concentré ou séparé, afin d'obtenir la multiplication des séquences-cibles spécifiques de l'oncogène. Ce procédé convient comme test universel de dépistage de malignité.
PCT/DE1988/000384 1987-06-29 1988-06-27 Procede d'analyse de fluides biologiques pour depister de l'adn cellulaire oncogene ou des fragments de celui-ci Ceased WO1989000206A1 (fr)

Applications Claiming Priority (2)

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DEP3721400.4 1987-06-29
DE19873721400 DE3721400A1 (de) 1987-06-29 1987-06-29 Verfahren zur untersuchung einer azellularen biologischen fluessigkeit auf zellulare onkogen-dna bzw. deren fragmente

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WO1989000206A1 true WO1989000206A1 (fr) 1989-01-12

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022456A1 (fr) * 1992-04-27 1993-11-11 Trustees Of Dartmouth College Detection de sequences geniques dans des liquides biologiques
US6020124A (en) * 1992-04-27 2000-02-01 Trustees Of Dartmouth College Detection of soluble gene sequences in biological fluids
US6156504A (en) * 1996-03-15 2000-12-05 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US6511805B1 (en) 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
US6630301B1 (en) 1997-03-14 2003-10-07 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
US8048629B2 (en) 1996-03-15 2011-11-01 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19736691A1 (de) * 1997-08-22 1999-02-25 Michael Prof Dr Med Giesing Verfahren zur Charakterisierung und Identifizierung disseminierter und metastasierter Krebszellen
RU2249820C1 (ru) * 2003-08-18 2005-04-10 Лактионов Павел Петрович Способ ранней диагностики заболеваний, связанных с нарушением функционирования генетического аппарата клетки

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EP0111340A2 (fr) * 1982-12-13 1984-06-20 Genzyme Corporation Procédé de liaison de l'acide nucléique à un support
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US5496699A (en) * 1992-04-27 1996-03-05 Trustees Of Darmouth College Detection of allele - specific mutagens
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US7282335B2 (en) 1996-03-15 2007-10-16 The Pennsylvania State University Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
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US6521409B1 (en) 1996-03-15 2003-02-18 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US8361726B2 (en) 1996-03-15 2013-01-29 The Penn State Research Foundation Method of detecting tumor-associated DNA in plasma or serum with a premalignant solid tumor
US6939675B2 (en) 1996-03-15 2005-09-06 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US7183053B2 (en) 1996-03-15 2007-02-27 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
US7208275B2 (en) 1996-03-15 2007-04-24 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US6156504A (en) * 1996-03-15 2000-12-05 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US7288380B1 (en) 1996-03-15 2007-10-30 The Pennsylvania State University Detection of extracellular translocated tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US6511805B1 (en) 1996-03-15 2003-01-28 The Penn State Research Foundation Methods for detecting papillomavirus DNA in blood plasma and serum
US7399592B2 (en) 1996-03-15 2008-07-15 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US7410764B2 (en) 1996-03-15 2008-08-12 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US7569349B2 (en) 1996-03-15 2009-08-04 The Penn State University Methods for selecting monoclonal antibody therapies using extracellular DNA
US7569350B2 (en) 1996-03-15 2009-08-04 The Penn State University Methods for selecting cancer vaccine therapies using extracellular DNA
USRE41327E1 (en) 1996-03-15 2010-05-11 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays
US7790395B2 (en) 1996-03-15 2010-09-07 The Penn State Research Foundation Evaluation of diseases and conditions associated with translocated genes using plasma or serum DNA
US7935487B2 (en) 1996-03-15 2011-05-03 The Penn State Research Foundation Method of detecting tumor-associated DNA in plasma or serum from humans without cancer
US7935484B2 (en) 1996-03-15 2011-05-03 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assay
US8048629B2 (en) 1996-03-15 2011-11-01 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
US6630301B1 (en) 1997-03-14 2003-10-07 The Penn State Research Foundation Detection of extracellular tumor-associated nucleic acid in blood plasma or serum

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EP0370032A1 (fr) 1990-05-30

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