EP1121467A2 - Test rapide pouvant etre automatise pour la detection directe de la mutation associee a la resistance induite par la proteine c activee (apc) au moyen d'amorces et de sondes specifiques - Google Patents

Test rapide pouvant etre automatise pour la detection directe de la mutation associee a la resistance induite par la proteine c activee (apc) au moyen d'amorces et de sondes specifiques

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Publication number
EP1121467A2
EP1121467A2 EP99950696A EP99950696A EP1121467A2 EP 1121467 A2 EP1121467 A2 EP 1121467A2 EP 99950696 A EP99950696 A EP 99950696A EP 99950696 A EP99950696 A EP 99950696A EP 1121467 A2 EP1121467 A2 EP 1121467A2
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EP
European Patent Office
Prior art keywords
starter
mutation
oligonucleotide
dna
amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99950696A
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German (de)
English (en)
Inventor
Wolfgang Springer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics GmbH Germany
Original Assignee
Bayer AG
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Filing date
Publication date
Application filed by Bayer AG filed Critical Bayer AG
Publication of EP1121467A2 publication Critical patent/EP1121467A2/fr
Withdrawn legal-status Critical Current

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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
    • 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 present invention relates to an automatable genetic test for the detection of the thrombosis risk factor APC (activated protein C) suitable starter oligonucleotides, oligonucleotide probes for this genetic test, as well as a method and a test kit for the detection of the APC resistance mutation.
  • APC activated protein C
  • thrombosis tendency can also be caused by a decrease in fibrinolytic activity in the form of an impaired release of t-PA (Grimaudo et al., Thrombos Haemostas, 67, 397, 1992) or an increase in the plaminogen activator.
  • Inhibitors (Engesser et al., Thrombos Haemostas, 62,673,1989) take place. However, no inheritance of these defects was observed.
  • factor V causes the development of APC resistance (Dahlbburg et al, PNAS 91, 1396, 1994).
  • factor V has the task of converting prothrombin to thrombin additionally an anticoagulant function. He is involved in the proteolytic inactivation of factor Va as a cofactor of activated protein C. Thrombin activation increases the procoagulatory effect of factor V.
  • the great advantage of the described invention is that by using special mutation- and wild-type-specific primers optimized with regard to length and mismatch sequences at the 3rd end, in contrast to the previously known methods based on amplification, no subsequent cleavage of the amplicon with a restriction enzyme is necessary and the detection of homozygous, heterozygous presence of the mutation or the wild type is not possible
  • RNA detector probe in conjunction with a DNA / RNA antibody, the signal strength of the amplicon could be increased by a factor of 10 and the sensitivity of the test compared to conventional DNA tests by a factor of 10 to 100.
  • the amount of test material required was greatly reduced and the reliability of the test statement was significantly improved by the significantly higher signals even with small amounts of test material. Thanks to the already developed automation of the process, it is possible to read out a large number of samples (> 100) within 20 minutes.
  • the present invention describes mutation-specific primers and oligonucleotide probes and their use for the rapid detection of APC resistance on the basis of mutation 1691 in exon 10 of the factor V gene in clinical sample material and biological body fluids without restriction enzyme cleavage and gel-electrophoretic identification of the cleavage products.
  • the amplification with the mutation primer or the wild type primer takes place only if the mutation or no mutation is present.
  • the method is particularly fast and automatable because the mutation type of the APC resistance can be determined directly via the amplification and in the readout, for example with magnetic beads according to the methods described in Examples 5 and 6, an automatic implementation is possible, e.g. Immuno I, Bayer Diagnostics, Tarrytown.
  • the invention relates to starter oligonucleotides containing the nucleotide sequence according to
  • the invention further relates to a set of the above-mentioned starter oligonucleotide and a starter nucleotide containing the nucleotide sequence according to the sequence listing SEQ ID No. 3. This set is suitable for the amplification and the specific detection of G / A point mutations in position 1691 of the nucleotide sequence of exon X from factor V Leiden gene.
  • the invention further relates to starter oligonucleotides containing the nucleotide sequence according to the sequence listing SEQ ID No.
  • the invention further relates to oligonucleotide probes, optionally labeled, containing the nucleotide sequence according to the sequence listing SEQ ID No. 4; Oligonucleotide probes, optionally marked, containing the nucleotide sequence according to
  • the invention relates to a method for the detection of APC gene sequences in a sample material, characterized in that
  • the invention further relates to a test kit for the detection of the APC resistance mutation containing one or more of the above-mentioned starter oligonucleotides and optionally one or more of the above-mentioned oligonucleotide probes.
  • the starter oligonucleotides were produced from the gene sequence of the factor V gene (Cripe, Biochemistry 1992, Genbank Acession Number J05368) by chemical synthesis.
  • the invention relates to primers and probes with a length of 15 to 50, preferably 18 to 30, particularly preferably 19 to 25 nucleotides from the region of the mutation 1691.
  • the primers carry the nucleotide of the mutation or of the wild type at the 3 'end and additionally several , preferably two to five, particularly preferably 2 further mismatch bases for better differentiation between wild type and mutation.
  • the preferred primers were selected from the range
  • the 1533-1616 range oligonucleotide probes were prepared by chemical synthesis (SEQ ID No. 4,5). This area is amplified by all primers and is specific for the factor V gene.
  • the oligonucleotide probes have a length of 20 to 100, preferably 20 to 40 nucleotides.
  • the genomic DNA was isolated from blood by nucleic acid isolation procedures.
  • the amplification of parts of exon 10 from factor V gene was carried out with the specific primers 1 or 2 from the coding region, which carry the base of the resistance mutation at the 3rd end (A instead of G (mutation primer) and G (wild type primer)) and a specific primer 3 from the non-coding strand of the gene, which is used as a forward primer for all amplifications.
  • a capture and detector sample from the nucleotide range 1533-1568 or 1581-1616 is preferably used . These are generally from 20 to 100 nucleotides in length, preferably from 20 to 50 nucleotides.
  • the amplification was carried out using known amplification techniques, preferably the PCR-DNA amplification method (EP200362).
  • the detection of the specific amplification product can be carried out according to known methods, for example by:
  • amplification product labeled with fluorescence nucleotides or fluorescence-labeled primers during the amplification.
  • the amplification product is separated using additional biotin (primer or nucleotide);
  • Amplification product performed with the above oligonucleotide probes.
  • the hybridization complex is separated with magnetic particles coated with fluorescein antibodies.
  • the evaluation of the hybridization complex formed as a measure of the amount or the presence or absence of the APC resistance mutation can done according to known methods.
  • the test is performed with a mutation primer approach and one approach
  • Wild-type primers carried out for the genotype differentiation of wild-type-specific, heterozygous or homozygous presence of the gene defect In the normal APC gene, amplification takes place only with the wild-type primer, the mutation primer does not give an amplicon and therefore no chemiluminescence signal. In the case of a heterozygous gene defect, an amplicon with both mutation primer and wild type primer is formed, and in the case of a homozygous gene defect, only the mutation primer gives an amplicon and chemiluminescence signal.
  • the amplification can be used to decide directly which gene defect is present, the test can be automated and, because of a single implementation step, is much faster and less labor and cost-intensive than conventional tests.
  • Gene probe diagnostics in particular in conjunction with amplification techniques, is a fast, specific and highly sensitive method that enables early detection of specific genes, gene fragments or individual mutations at the DNA / RNA level.
  • the technique can be carried out directly in the test material. It is based on the DNA / RNA hybridization technique, ie the specific in vitro binding of complementary single-stranded nucleic acid with the formation of Watson-Crick base pairs.
  • the DNA / DNA or DNA / RNA double strands formed are also referred to as DNA hybrids.
  • Complementary sequence-specific gene probes are used to detect the specific DNA or RNA by means of the hybridization reaction. These gene probes are short, chemically synthetic tized oligonucleotide probes with a length of 10 to 200, preferably 18 to 35, nucleotides.
  • the gene probes can be photochemically (N.Dattagupta, PMMRae, ED Huguenel, E. Carlson, A. Lyga, JS Shapiro, JP Albarella, Analytical Biochem. 177.85.1989) or enzymatically by nick translation (Rigby, PWJ et al., J .Mol.Biol.
  • Biochem. 132.6.1983) with a radioactive or non-radioactive label with a radioactive or non-radioactive label.
  • Suitable for this are labels with 3 PNTPs or non-radioactive labels with reporter molecules such as digoxigeninide-UTP, biotin-DUTP or direct labeling with enzymes such as alk. Phosphatase or Horseradish
  • the nucleic acids are first denatured
  • the gene probe only binds to complementary sequences of the DNA or RNA to be detected.
  • This hybridization reaction can be carried out in various test formats e.g. as solid phases on a carrier such as e.g. Nitrocellulose-coupled target DNA or gene probe or as a liquid hybridization.
  • the evaluation takes place via the labeling of the gene probe with a reporter molecule as listed above or, as in the reversed phase hybridization system shown here, via the target DNA, which occurs during the
  • Amplification is labeled with digoxigenin-dUTP and the gene probe that is labeled with biotin for binding to magnetic particles.
  • the hybridization complex of target DNA and labeled gene probe is determined quantitatively after the removal of unbound gene probe via the reporter molecule used. This read out can be done directly with fluorescence labeling or radioactive
  • Antibody conjugates the enzymes such as the alk. Contain phosphatase and then allow a color reaction or chemiluminescence reaction.
  • test sensitivity with the gene probe diagnosis is in the range of 10 5 to 10 6 copies based on the detection of single genes.
  • An increase in test sensitivity can be achieved by combining it with DNA or RNA amplification techniques such as PCR (EP 200362). LCR (EP 320308), NASBA (EP 329822), Qß (PCT 87/06270) or HAS technology (EP 427074) can be achieved. With these techniques, up to 10 9- fold multiplication of the DNA to be detected can be achieved. The combination of amplification and hybridization makes it possible to detect individual DNA molecules.
  • Suitable specific primers were selected on the basis of the nucleotide sequence of exon 10 of the factor V Leiden gene (Cripe, Biochemistry 1992, Genbank Acession Number J05268). On the basis of the mutation sequence and the wild-type nucleotide sequence, primers were selected which carry the base of the point mutation at the 3rd end. For the G / A mutation type, the primer SEQ ID No. 1 synthesized. The primer 2 SEQ ID No. 2 synthesized. For the
  • primer 3 SEQ ID No. 3 of which is common to all primer sets (primers 1 + 3, 2 + 3).
  • the primers additionally carry several, preferably two further mismatch bases, for better differentiation between wild type and mutation.
  • the selected primers were chemically synthesized using the phosphoramide method of SL Beaucage and M.Caruthers, Tetrahedron Letters, 22.1859.1981. Amplification of genomic DNA
  • primer set 1 primer 1 + 3
  • primer set 2 primer 2 + 3
  • Digoxigenin-dUTP can be incorporated into the amplification product.
  • an antidigoxigenin antibody e.g. alk.
  • fluorescence-labeled nucleoside triphosphates such as e.g. Incorporate fluorescein dUTP or coumarin dUTPs into the amplification product and identify the amplification product with much higher sensitivity than with ethidium bromidan staining.
  • biotinylated primers it is possible to separate the fluorescence-labeled, biotinylated amplification product via streptavidin-coated magnetic particles and to determine them quantitatively in the fluorescence photometer.
  • streptavidin-coated magnetic particles it is possible to separate the fluorescence-labeled, biotinylated amplification product via streptavidin-coated magnetic particles and to determine them quantitatively in the fluorescence photometer.
  • a DNA capture probe and an unlabeled RNA probe are used as the detector probe and the read out is carried out using a DNA / RNA antibody. It is also possible to use only one sample in the form of a fluorescein-labeled RNA sample, which serves as a capture and detector sample. With this new genetic test using the DNA / RNA antibody, significantly better sensitivities than with the previously usual genetic tests for other targets achieved and therefore very little starting material needed to carry out the test.
  • the oligonucleotide probes specific for the amplification products of the primer sets were selected from the range 8167-8486 for which the mutation-specific and wild-type-specific amplification product is common. 30-36 primers with the sequence SEQ ID No. 4 and 5, which are specific for the two amplification products. Alternatively for SEQ ID No. 4 can SEQ ID No.
  • oligonucleotide probes it is possible to specifically hybridize the mutation-specific and wild-type-specific amplification products.
  • the method in which parts of the exon 10 of the factor V Leiden gene are first amplified with the mutation-specific primers and then the amplification product is then specifically hybridized with the two oligonucleotide probes has the advantage over pure amplification diagnostics via the gel that the test - sensitivity is higher by a factor of 100-1000 and the time-consuming and labor-intensive process cannot be automated:
  • the APC mutation can be determined directly after amplification of a part of the exon 10 gene from Factor V Leiden by the primer sets described in the invention using any analytical method.
  • a possible read out method is the staining of the amplification product separated by agarose gel electrophoresis with intercalating agents such as ethidium bromide.
  • fluorescence-labeled primers for the amplification or the combination of biotinylated primers with fluorescence nucleotides, so that a terminally biotinylated, fluorescence-labeled amplification product is formed, which can be bound and separated to streptavidin-coupled magnetic particles and the fluorescence can be determined semiquantitatively can.
  • the most sensitive and preferred method is the described method of hybridizing the amplification products with the described oligonucleotide probe. If, for example, digoxigenin-dUTP is incorporated during the amplification and use of a biotinylated or fluorescent oligonucleotide probe, the hybridization complex of streptavidin / fluorescein antibody-coated magnetic particles can be separated and when using antigigoxigenin antibodies which are used with alk. Phosphatase are coupled, with AMPPD or CSPD as a subsrate evaluate semiquantitatively via chemiluminescence.
  • the preferred method is the amplification without any incorporation of marker molecules and the detection of the amplicon by hybridization with a fluoresein-labeled capture probe and an additional RNA probe as a detector sample.
  • the hybridized amplicon is detected using a DNA / RNA antibody. This read out results in the highest sensitivity of all test methods tested so far and was specially developed for automated methods, for example in immunol machines and successor devices. example 1
  • the selected starter oligonucleotides were chemically synthesized using the phosphoramidite method of S.L. Beaucage and M.Caruthers, Tetrahedron Letters, 22.1859.1981.
  • the following nucleotide sequences were synthesized:
  • PCR primer 1 specific f APC mutation: SEQ ID No. 1 PCR primer 2 specific f. Wild type APC: SEQ ID No. 2
  • the oligonucleotide probes were selected from the nucleotide region 8167-8486 of exon 10 of the factor V Leiden gene, which contains the mutation-relevant region.
  • the selected oligonucleotide probes were chemically synthesized using the phosphoramidite method of S.L. Beaucage and
  • TACTTATAAGTGGAACATCTTAGAGTTTGATGAACC Capture Probe SEQ ID No. 5
  • TGCCCAGTGCTTAACAAGACCATACTACAGTGACGT RNA probes SEQ ID No. 6-7
  • the detector probe was labeled at the 3rd end using the method of Bollum, The enzymes, Bayer ed, Vol. 10, p 145 Academic Press New York. End group labeling was not radioactive with fluorescein dUTP (Chang, L.M.S., Bollum T.J., J. Biol. Chem. 246.909.1971). In a 50 ml batch with 10 ml
  • Reacton buffer (potassium cocodylate 1 mol / 1; Tris / HCl 125 mmol / l; bovine serum albumin 125 mg / ml; pH 6.6; 25 ° C) 1 to 2 mg oligonucleotide, 25 units terminal transferase CoCl 2 2.5 mmol / 1 and 1 ml fluorescein-dUTP (1 mmol / 1) is reached after 60 minutes at 37 ° C approx. 50% 3rd end label.
  • the detector probe was labeled analogously with digoxigenin-dUTP or an unlabeled RNA sample was used.
  • the amplification of the target DNA was carried out after the polymerase chain reaction (EP 200362; 201184).
  • 100 pg of genomic DNA from blood cells (leucocytes), 0.17 ⁇ mol primer 1-5 SEQ ID No. were used for the PCR reaction.
  • 1-5 2.5 units of Taq polymerase from Cetus / Perkin-Elmer and 200 ⁇ mol / 1 dNTPS in a total of 100 ⁇ l PCR buffer (50 mM KC1, 10 mM Tris / HCl pH 8.3, 2 mM MgCl 2 , and 0.01% gelatin.
  • the amplification was carried out in a PCR processor from Cetus / Perkin-Elmer.
  • a total of 2 PCR batches with primer set 1 (primer 1 + 3) and primer set 2 (primer 2 + 3 for detection of homozygous, heterozygous or wild-type APC gene is set in.
  • An amplification signal with wild-type + mutation primer shows the heterozygous presence of the Mutation
  • a signal with wild type but not with mutation primers shows the wild type
  • a signal with the mutation primer set but not with the wild type primer shows the homozygous presence of the mutation.
  • RNA probe is used as a detector probe in conjunction with a DNA / RNA antibody (example 7), the PCR is carried out without labeling additives.
  • the plasmid DNA must be diluted 1:10 5 before the PCR. From this dilution 1 ⁇ l is then used and 99 ⁇ l PCR mix. This corresponds to a concentration of 100 ng clinical sample. The clinical sample is used in a concentration of 100 ng, also 1 ⁇ l DNA and 99 ⁇ l mix. PCR mix, 10-fold: 100 ⁇ l 10 x PCR buffer
  • intercalating agents such as e.g. Ethidium bromide or fluorescence nucleotide triphosphates such as e.g. Fluorescein dUTP / or coumarin dUTP incorporated.
  • Biotin-dUTP or digoxigenin-dUTP can also be used, and alk. Phosphatase a dye read out can be performed.
  • fluorescence-labeled primers can also be used with a lower sensitivity.
  • the preferred method was the incorporation of coumarin-UTP because the best test sensitivity was achieved.
  • the amplification product was applied to a 0.8% agarose gel and electrophoresed at 100 mA for 30 minutes. The fluorescence signals were evaluated directly under a UV transilluminator.
  • PCR Polymerase Chain Reaction
  • LCR LCR
  • gene probe technology a significant increase in sensitivity compared to conventional gene probe readout methods is achieved.
  • liquid hybridization tests were carried out with 100 ng of 3'-biotinylated or digoxygenated detector probe and fluorescent capture probe and amplified DNA according to Example 3 in a volume of 50 ⁇ l.
  • Buffer B (0.1 SSC; 0.1% SDS) washed 1x.
  • the blocking reaction and antibody reaction for detection of hybridization via chemiluminescence was then carried out.
  • the beads loaded with DNA were washed 1x with 500 ⁇ l blocking buffer (0.1 M maleic acid; 0.15 M NaCl pH
  • PCR polymerase chain reaction
  • LCR LCR
  • the liquid hybridization tests were carried out with 100 ng of 3 'unlabeled RNA detector probe and fluorescent capture probe and amplified DNA according to Example 3 in a volume of 50 ⁇ l. After heating for 5 minutes at 100 ° C, 50 ⁇ l 2x hybridization mix (50 ml 20XSSC, 1g blocking reagent (Boehringer), 2 ml 10% lauroyl sarcosine, 200 ml 20% SDS ad 100 ml Bidest H 2 O) were added and 5 hybridized up to 10 minutes at 37 ° C (oligonucleotide probe).
  • 50 ⁇ l 2x hybridization mix 50 ml 20XSSC, 1g blocking reagent (Boehringer), 2 ml 10% lauroyl sarcosine, 200 ml 20% SDS ad 100 ml Bidest H 2 O
  • the magnetic beads (20 ⁇ l) are added to the hybridization mixture and 100 ⁇ l lx hybrid mix and incubated for 5 to 10 minutes at room temperature with gentle agitation.
  • the coupled hybridization complex was separated with the beads, the residual liquid was pipetted off and washed once with buffer B (0.1 SSC; 0.1% SDS) once.
  • the blocking reaction and antibody reaction for detection of hybridization via chemiluminescence was then carried out.
  • the beads loaded with the DNA-RNA hybrid were added 1x with 500 ⁇ l blocking buffer (0.1 M maleic acid; 0.15 M NaCl pH 7.5; 1% blocking reagent (Boehringer)). After 5 minutes of incubation at room temperature, the mixture was separated, pipetted off and 250 ⁇ l of antibody conjugate solution (AK 1: 2500 in blocking buffer) were added and 10
  • Chemiluminescence measured for 10 seconds in the luminescence photometer at 477 nm (Lumaco below from Lumac). The process can be carried out automatically on the Immunol or subsequent devices.
  • protease stock solution After the addition of 25 ⁇ l of protease stock solution, the mixture is incubated at 50 ° C. for 30 minutes and applied to the Qia column with 1 ml of QBT buffer and eluted. The Qiagen column is washed with 3x 1 ml buffer QC and then the genomic DNA is eluted with 2x 1 ml buffer QF. After adding 0.7 ml of isopropanol at 15 ° C 15
  • the leucocyte DNA was isolated from the clinical sample material (blood) according to the method described in Example 7.
  • the DNA lysate was then amplified with the aid of suitable amplification methods as described in Example 5 with specific oligonucleotide primers.
  • the amplified nucleic acid was then analyzed with the oligonucleotide probes SEQ ID No. 4 + 5 hybridized and the under stringent

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Abstract

La présente invention concerne un test génétique pouvant être automatisé, permettant de détecter le facteur de risque thrombotique APC (protéine C activée), des oligonucléotides de départ appropriés et des sondes oligonucléotidiques pour ce test génétique, ainsi qu'un procédé et une trousse de test pour la détection de la mutation associée à la résistance induite par APC.
EP99950696A 1998-10-22 1999-10-11 Test rapide pouvant etre automatise pour la detection directe de la mutation associee a la resistance induite par la proteine c activee (apc) au moyen d'amorces et de sondes specifiques Withdrawn EP1121467A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19848665 1998-10-22
DE19848665A DE19848665A1 (de) 1998-10-22 1998-10-22 Ein automatisierbarer Schnelltest zum direkten Nachweis der APC Resistenz Mutation mit spezifischen Primern und Probes
PCT/EP1999/007609 WO2000024928A2 (fr) 1998-10-22 1999-10-11 Test rapide pouvant etre automatise pour la detection directe de la mutation associee a la resistance induite par la proteine c activee (apc) au moyen d'amorces et de sondes specifiques

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EP1121467A2 true EP1121467A2 (fr) 2001-08-08

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EP99950696A Withdrawn EP1121467A2 (fr) 1998-10-22 1999-10-11 Test rapide pouvant etre automatise pour la detection directe de la mutation associee a la resistance induite par la proteine c activee (apc) au moyen d'amorces et de sondes specifiques

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EP (1) EP1121467A2 (fr)
AU (1) AU6337699A (fr)
DE (1) DE19848665A1 (fr)
WO (1) WO2000024928A2 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES8706823A1 (es) * 1985-03-28 1987-06-16 Cetus Corp Un procedimiento para detectar la presencia o ausencia de al menos una secuencia especifica de acidos nucleicos en una muestra
DE69231734T2 (de) * 1991-01-16 2001-09-20 Univ Johns Hopkins Geerbte und somatische mutationen des apc-gens bei menschlichem colorectal-krebs
JP3163105B2 (ja) * 1994-02-14 2001-05-08 リヤクス・ユニフアーシテイト・レイデン 血栓症及び／又は活性化プロテインｃに対する弱い抗凝血応答に関連した遺伝性欠陥の存在をスクリーニングする方法
CA2216239A1 (fr) * 1995-03-24 1996-10-03 The Scripps Research Institute Methodes d'identification d'une mutation de genes de facteurs v
AT403289B (de) * 1996-01-30 1997-12-29 Immuno Ag Verfahren zur herstellung eines standardisierten plasmapools für diagnostische zwecke
US5879890A (en) * 1997-01-31 1999-03-09 The Johns Hopkins University APC mutation associated with familial colorectal cancer in Ashkenazi jews

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0024928A3 *

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AU6337699A (en) 2000-05-15
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WO2000024928A3 (fr) 2000-06-22

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