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WO1992019775A1 - Titrage et lot d'epreuve pour le depistage des anomalies chromosomiques - Google Patents

Titrage et lot d'epreuve pour le depistage des anomalies chromosomiques Download PDF

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Publication number
WO1992019775A1
WO1992019775A1 PCT/EP1992/000929 EP9200929W WO9219775A1 WO 1992019775 A1 WO1992019775 A1 WO 1992019775A1 EP 9200929 W EP9200929 W EP 9200929W WO 9219775 A1 WO9219775 A1 WO 9219775A1
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WIPO (PCT)
Prior art keywords
process according
sequence
target sequence
probes
translocation
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PCT/EP1992/000929
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English (en)
Inventor
Giorgio Martinazzo
Roberta Bichi
Stanislavo Marcolini
Elisabetta Turchetti
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Raggio-Italgene Spa
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Publication date
Application filed by Raggio-Italgene Spa filed Critical Raggio-Italgene Spa
Priority to AU16499/92A priority Critical patent/AU666648B2/en
Priority to JP4508516A priority patent/JPH06509464A/ja
Publication of WO1992019775A1 publication Critical patent/WO1992019775A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to a process and kit for use in detecting the nucleic acid sequences that occur in chromosomal abnormalities.
  • Chromosomal abnormalities are the cause of various undesirable conditions in humans, both inherited and non- inherited, including neoplastic conditions such as follicular lymphoma. It is clearly of vital importance to detect such abnormalities, whether caused by chromosomal translocation, transposition, intergene or intragene recombination, insertion, deletion or point mutation at an early stage by a simple and reliable test.
  • translocations and pathological states e.g. neoplastic degeneration
  • Russo _____ al in "Recent Advances in He atology", A.V. Hoffbrand (ed.), 5, 121-130, Churchill Livingstone. More particularly, a translocation t(14;18) that involves portions of the genes bcl-2 and J H has been strongly correlated to human follicular lymphoma; see Tsujimoto et a_l (1985) Science 228: 1440-1443, and Science 229: 1390-1393; Stetler-Stevenson et al (1988) Blood 7_2: 1822-1825; and Crescenzi et a_l (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 4869-4873.
  • Follicular lymphoma is a B-cell disorder which is related to the presence of a genetic abnormality called the bcl-2 translocation.
  • About 90% of follicular B-cell lymphomas and 20% of large diffuse B-cell lymphomas carry the t(14;18) (q32;q21) translocation which directly involves the IgH locus on chromosome 14 and the bcl-2 locus on chromosome 18.
  • the t(14;18) (q32;q21) translocation occurs 5' or 3' to the bcl-2 gene, but not within the protein coding portion of the gene. It appears that in FL the translocation takes place in pre-B-cells during the
  • the FL translocations are structurally uniform. In about 70% of human FL the breakpoints are clustered within the 3 1 untranslated region of the gene, designated “Major Breakpoint Region” (MBR) . In another 10-20% of the cases, the breakpoints are clustered in a region more than 20 Kb downstream from bcl-2's second exon, designated “minor cluster region” (mcr) . In some cases translocations have been detected near the 5' exon.
  • GB-A-2169403 describes a method for the identification of nucleic acids, in which two independently-rlabelled oligonucleotide probes are reacted in a single solution, under hybridising conditions, with a target analyte. If the analyte contains a sequence that hybridises to both probes, this may readily be detected by virtue of the fact that one label allows separation of the hybrid and the other its detection.
  • EP-A-0128332 EP-A-0145356, EP-
  • a process of the general type described in GB-A-2169403 is applied to the detection of chromosomal abnormalities, e.g. translocations, using capture and reporter probes that are respectively complementary to different regions of the target sequence, e.g. on opposite sides of the translocation.
  • the respective probes, and any other components used in the procedure, as required, may be formulated into a novel kit comprising a plurality of containers in which the components are distributed.
  • the present invention provides a number of valuable characteristics. Firstly, for example, it is simple to use, e.g. by relatively unskilled personnel in hospitals and less specialised laboratories; it is quick, non-radioactive and requires only simple equipment.
  • the absorbance readings allow a quantitative measurement of the final signal.
  • this quantitative determination of signal is only possible with the use of sophisticated instrumentation. These traditional methods are much more prone to subjective interpretation.
  • the quantitation of the signal allows much easier comparison of results between experiments, and between laboratories.
  • the system will only generate a signal if both reporter and capture probes (complementary to sequences on either side of the breakpoint) bind. This provides a very high degree of specificity and helps minimise the risk of false positives; this is particularly important as this technology has primarily been designed to detect chromosomal translocations associated with malignancies.
  • the nucleic acids in the analyte preferably comprise double-stranded DNA. They may be amplified by the action of DNA polymerase which is capable of synthesising in the 5'-3' direction a complementary strand from a template, in the presence of a primer which is complementary to an extreme portion of the single-stranded analyte sequence. Preferably, amplification occurs for both strands of the analyte sequence, and the DNA polymerase is heat-stable. The amplified strands may then be denatured.
  • the denaturation occurs by means of incubation, e.g. at a temperature between 90 and 97 P C, or in the presence of NaOH.
  • the capture probe is conjugated to a hapten such as fluorescein isothiocyanate (FITC) .
  • FITC fluorescein isothiocyanate
  • separation is by means of anti-hapten antibodies, e.g. anti-FITC, which are immobilised on a solid phase, preferably magnetisable microparticles which are attracted onto magnetic plates.
  • the liquid phase containing free detection probes may be removed by washing.
  • the detection probe is preferably conjugated to an enzyme or biotin. Detection is then conducted by means of incubation with a substrate which is specific for the enzyme, preferably chro ogenic, termination of the reaction, e.g. by adding a stop solution, and colorimetric reading of the solution itself.
  • the enzyme is an alkaline phosphatase
  • the specific chromogenic substrate is phenolphthalein monophosphate
  • ET reading is carried out at a wavelength of 554 nm.
  • a probe that is conjugated to biotin may be detected by means of avidin conjugated to an enzyme.
  • the present invention is particularly useful for the detection of nucleic acid sequences comprising contiguous DNA segments from different chromosomes, or from different zones of the same chromosome. This may be the result of any of the following biological processes: chromosomal translocation, transposition, intergene or intragene recombination, insertion, deletion or point mutation.
  • the translocation may be correlated to a neoplastic state, as for instance those related to T and B lymphocytes; for example, the translocation may be t(14;18), the analyte sequence bcl- 2/J H , and the neoplastic state follicular lymphoma.
  • the sequence of the analyte DNA contains the recombination point of two human chromosomes 14 and 18, and the probes bind to either side of the target sequence on the same DNA strand, e.g. the negative strand.
  • the primers be of such length and composition as not to allow hybridisation to occur with themselves or with portions of the analyte DNA segment which is complementary to the other primer. Accordingly, the extension products are synthesised employing a DNA polymerase, which is preferably heat-stable, and extends the terminal portion to the 3' position of each primer.
  • extension products are then separated from their templates by means of high temperature denaturation (92- 94°C) .
  • the passage is repeated through a number of cycles sufficient to increase the amount of the target sequence up to the concentration at which it can be detected.
  • a suitable amount of the analyte sequence is caused to react with a suitable amount of the analyte sequence.
  • denaturation can be carried out through exposure of the DNA to a temperature of 94-97°C for 5-10 minutes and then cooling suddenly down to 0°C.
  • a second pair of oligonucleotides is employed. These are probes which are different from the primers employed in the amplification procedure and which are both complementary to the same strand of the analyte DNA, in zones which are to those employed for amplification.
  • the probes are added to the reaction mixture at an excess concentration with respect to the analyte sequence.
  • the pair of probes consists of a capture oligonucleotide and of a reporter oligonucleotide. Each probe is conjugated through its 5 ' end with a reactive group, to provide an appropriate label.
  • Reper molecules include haptens, enzymes and radioactive labels, or include any substrate that provides a chromogenic, fluorescent or chemiluminescent signal.
  • the report probe is labelled with alkaline phosphatase and the capture probe with a hapten such as FITC.
  • the capture probe is suitably separated by linkage to a solid phase such as plastics beads, microplates, coated tubes, latex or, preferably, magnetisable microparticles.
  • a hapten can be linked specifically by an antibody immobilised on a solid phase, e.g. anti-FITC on magnetisable microparticles.
  • a neutralising solution e.g. 0.5 M Tris, pH 7.5, is added to the reaction mixture, in such an amount as to buffer the NaOH and allow the hybridisation of the probes to the analyte DNA to occur.
  • SUBSTITUTE SHEET probe both the free and that reacted with the DNA sequence, is added to the reaction mixture, so forming the analyte sequence-probes complex.
  • a suitable incubation period at a constant temperature e.g. 10 minutes at +37°C
  • the reaction tubes are put on a magnetic plate which, in a short time, e.g. 3 minutes, causes the magnetisable particles to settle onto the bottom of the tube itself.
  • the supernatant is then removed by decantation, by turning the magnetic plate upside down, the magnetised particles adhering to the bottom of the tube.
  • the washing cycle is repeated as often as is necessary to remove any non-specific binding of the reagents, and in particular of the reporter probe which is conjugated to the enzyme, with the solid phase.
  • all those reactants which are not specifically linked to the magnetic particles are removed from the reaction tube.
  • a suitable amount of a chromogenic substrate which is enzyme-specific e.g. 200 ⁇ l of phenolphthalein monophosphate, is added to the magnetic particles and allowed to react for the time required at a constant temperature, e.g. 1 hour at 37°C. After this period, the reaction is stopped by adding a stop solution, e.g. 750 ⁇ l of a Na 2 C0 3 solution, pH 12.
  • the addition of the stop solution causes the. formation and the stabilisation of colour, the absorbance value of which is measured at a suitable wavelength, for instance 554 nm, on a colorimeter.
  • a colour development which is significantly higher than that of blank samples indicates that, during amplification, some extension products were formed starting from the specific primers and from the analyte DNA sequence that has acted as a template. In the absence of the analyte sequence, no formation of specific
  • SUBSTITUTE SHEET extension products would have occurred, which products are the only compounds capable of acting as bridges between the magnetic particles and the reporter probe that bears the enzyme capable of generating the signal.
  • a standard curve may be generated, employing known concentrations of the analyte DNA, to give a concentration value for each sample analysed.
  • the method for conjugating a reactive group to the oligonucleotide probes obviously depends on the group type that is to be employed; generally the preferred bond occurs through the OH group in the 5* position of the oligonucleotide.
  • the oligonucleotide employing phosphoroamidite chemistry, it is possible to introduce an aliphatic amine at the 5* end employing the Aminolink 2 (ABI) reactant or the Aminomodifier II (Clontech) reactant; this a ino group can be reacted successively with a specific hapten, for instance FITC or a biotin-hydroxy-succinimide ester, or any other group containing an ester which is activated and capable of reacting with a primary amine.
  • a specific hapten for instance FITC or a biotin-hydroxy-succinimide ester, or any other group containing an ester which is activated and capable of reacting with a primary amine.
  • heterobifunctional reactants such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • SMCC 2-iminothiolane
  • 2-IT 2-iminothiolane
  • SMCC is capable of reacting with the primary amine in the 5' position of the reporter probe give a derivative having a maleimido group free
  • the 2-IT is capable of reacting with the NH 2 groups of lysines of the alkaline phosphatase so as to give a derivative, having a free -SH group.
  • the maleimido groups and the -SH group if caused to react under suitable conditions, react spontaneously so as to form a very stable carbon-sulphur covalent bond. In this way, it is possible to obtain conjugates in which the reporter probe is linked through its 5' end to the alkaline phosphatase through a long and flexible carbon atom chain, keeping the oligonucleotide capability of specifically hybridising with a complementary
  • SUBSTITUTE SHEET sequence unaltered, and keeping also unaltered the capability of the enzyme to interact with its specific substrate, to generate a coloured solution.
  • Magnetisable particles coated with anti-FITC antibodies are commercially available (from Ares-Serono,
  • the extension products can be generated by the exposure of the primers, hybridised to their templates, to a DNA polymerase which is preferably heat-stable, e.g. the
  • Taq polymerase disclosed ' in EP-A-0258017.
  • the DNA polymerase will replicate the sequence of the template, so synthesising some fresh DNA from the primers in the 5'-3' direction.
  • a heat-stable polymerase is preferred, but it is not indispensable because the simplest way of denaturing the double-stranded extension product is by exposure to high temperatures (about 95°C) during the cycles of the PCR, as disclosed in US-A-4683202.
  • other polymerases can be used, including the Klenow fragment.
  • MBR region on chromosome 18 (within the 3' untranslated region of the bcl-2 gene) ; and (iii) the mcr region on chromosome 18 in a region more than 20 Kb downstream from the bcl-2 second exon.
  • Preferred primers of these types which do not interfere with each other and which yield the same efficiency of amplification for both the MBR and the mcr sequences, are the J H primer shown as SEQ ID. No. 3,
  • MBR primer shown as SEQ ID No. 1 the MBR primer shown as SEQ ID No. 1
  • mcr primer shown as SEQ ID No. 2 see Sequence Listing, below.
  • either the MBR or the mcr- amplified sequence can be detected using specific reporters.
  • specific reporters it is known that six J H regions are present in 'the IgH locus.
  • a mixture of six modified oligonucleotides is preferably used. Each oligonucleotide is complementary to one of the six specific J H regions; they have the respective sequences shown as SEQ ID Nos. 4-9.
  • the FITC-conjugated oligonucleotide acts as a capture probe, because it reacts with the anti-FITC coated magnetic particles during the detection assay.
  • the mixture of the six conjugated oligonucleotides is used in the detection of both MBR and mcr-amplified sequences. The determination of which breakpoint is present is made possible by the use of specific probes for either the MBR or the mcr region of the bcl-2 gene.
  • Both the MBR and mcr reporter oligonucleotides are modified at both the 3 ' and 5' end with a NH- group during the automated synthesis. They are then conjugated to the enzyme alkaline phosphatase and purified as described above. The enzyme-conjugated oligonucleotides act as "signal generating" probes.
  • the modified oligonucleotides used as reporters (after being conjugated to alkaline phosphatase) are shown as SEQ ID NO: 1
  • reaction buffers in which either the MBR or the mcr detection probes are dissolved differ slightly from each other, in order to account for the different lengths of the amplified sequences (200 bp for the MBR and 400 bp for the mcr) .
  • the initial dilution of the PCR samples may vary for the two targets (for example, actual dilution for the MBR is 1:10; for the mcr 1:4) , as does the incubation time for the hybridisation step (for example, 30 minutes at +37°C for MBR and 15 minutes at +37°C for mcr) .
  • kits illustrate a kit of the invention and the "Recommended Procedures” illustrate the process of the invention. These are taken from the instructions associated with a kit marketed under the trade name C-TRAK FL by Raggio-Italgene S.p.A.
  • This kit is specifically designed for in vitro research use, for the detection of the t(14;18) (q32;q21) chromosomal translocation in frozen biopsies, paraffin- embedded tissues, peripheral blood and bone marrow. Prior DNA isolation can be conducted by standard methods, as described by Maniatis _____ _____ in "Molecular Cloning, A Laboratory Manual", 2nd ed. pub. Cold Spring Harbour.
  • Each kit contains sufficient PCR primers to run 25 amplifications. These amplifications can be subdivided into a maximum of 5 runs - 3 samples plus 2 PCR controls in each run.
  • JH-FITC reporters JHI-6 probes conjugated to FITC
  • a bcl-2- MBR probe conjuggated to the enzyme alkaline phosphatase
  • the recommended protocol for the Perkin-Elmer thermocycler 9600 includes the use of:
  • SUBSTITUTE SHEET 12 Dispense 0.1ml of thoroughly mixed Separation Reagent (reagent No. 6) into each tube.
  • the magnetic antibody suspension must be thoroughly mixed before use to ensure a uniform suspension of magnetic particles. After pipetting into 10 to 20 tubes swirl the vial.
  • Stop Solution it is critical to add Stop Solution at approximately the same rate and in the same sequence, as when adding the Substrate Solution.
  • Samples for which the absorbance exceeds the upper limit of the spectrophotometer should be read at 492nm.
  • A55 0 is approximately equal to 5 x A 4 92 , though the precise relationship should be determined for each instrument.
  • the A550 of the Negative PCR Control should not be significantly different from the A550 of the Negative Detection Control. If the A 550 of the Negative PCR Control does significantly exceed the A550 of the Negative Detection Control [i.e. (AC55 0 - 3 S.D.) > (Adsso + 3 S.D.), where d denotes the Negative Detection Control], then the results of the whole test run should be disregarded and actions implemented to avoid further PCR carry-over.
  • both the PCR Positive control and the Detection Positive Control must yield A55 0 values within the range indicated in the lot-specific data sheet provided with each kit.
  • translocation assay In our laboratories we have been able to detect the presence of 1 translocated cell in 50,000 cells. A negative result in the translocation assay could occur simply as a result of very low concentrations of translocated cells in the sample. Additionally, when investigating potentially low level occurrence of the t(14;18) translocation, statistical sampling methods should be employed.
  • Intra-assay precision of the detection step was determined by measuring A550 replicates of the same PCR amplified samples and resulted in an average CV of 8-10%.
  • Sequence Type Oligonucleotide Sequence Length: 21 bases Strandedness: Single Topology: Linear GAT GGC TTT GCT GAG AGG TAT
  • Sequence Type Oligonucleotide Sequence Length: 20 bases Strandedness: Single Topology: Linear ACC TGA GGA GAC GGT GAC CA
  • Sequence Type Oligonucleotide Sequence Length: 27 bases Strandedness: Single Topology: Linear NH 2 -GGT ACT TCG ATC TCT GGG GCC GTG GCA-NH 2

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Abstract

Séquence cible incluant une translocation chromosomique ou une autre anomalie, dépistée par réaction de ladite séquence cible dans des conditions d'hybridation en phase homogène avec une abondance de sondes oligonucléotides de capture et rapporteuses respectivement complémentaires de différentes régions de la séquence cible; tout hybride résultant présentant les deux marques est ainsi dépisté et séparé.
PCT/EP1992/000929 1991-04-29 1992-04-29 Titrage et lot d'epreuve pour le depistage des anomalies chromosomiques WO1992019775A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU16499/92A AU666648B2 (en) 1991-04-29 1992-04-29 Assay and kit for the detection of chromosomal abnormalities
JP4508516A JPH06509464A (ja) 1991-04-29 1992-04-29 染色体異常の検出用アッセイおよびキット

Applications Claiming Priority (2)

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IT286A/91 1991-04-29
ITRM910286A IT1244983B (it) 1991-04-29 1991-04-29 Procedimento per rivelare sequenze di acidi nucleici e kit per la sua utilizzazione.

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JP (1) JPH06509464A (fr)
AU (1) AU666648B2 (fr)
CA (1) CA2109518A1 (fr)
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WO (1) WO1992019775A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011990A1 (fr) * 1993-10-29 1995-05-04 Raggio-Italgene S.P.A. Dosage destine a la detection d'anomalies genetiques
US5595869A (en) * 1986-07-09 1997-01-21 The Wistar Institute Diagnostic methods for detecting lymphomas in humans
GB2311132A (en) * 1996-03-15 1997-09-17 Univ Heidelberg Labelled nucleic acid sequences which are specific for translocation breaking points
EP0805215A3 (fr) * 1996-04-30 1999-05-26 Vysis, Inc. Procédé et sondes diagnostiques
US5985598A (en) * 1994-10-27 1999-11-16 Thomas Jefferson University TCL-1 gene and protein and related methods and compositions
WO2003018835A3 (fr) * 2001-08-23 2004-03-25 Hvidovre Hospital Procede de detection rapide d'haplotypes
EP1544308A1 (fr) * 2003-12-15 2005-06-22 Institut Pasteur Détermination du répertoire d'une population de lymphocytes B
WO2005059176A1 (fr) * 2003-12-15 2005-06-30 Institut Pasteur Determination du repertoire d'une population de lymphocytes b
US7175995B1 (en) 1994-10-27 2007-02-13 Thomas Jefferson University TCL-1 protein and related methods
CZ302581B6 (cs) * 2010-06-04 2011-07-20 Masarykova Univerzita Zpusob detekce chromozomální translokace t(11;14)(q13;q32) a oligonukleotidy pro použití pri tomto zpusobu
US8034555B2 (en) * 1999-10-08 2011-10-11 University Of Utah Research Foundation Particle analysis assay for biomolecular quantification
US8105608B2 (en) 2000-03-31 2012-01-31 Purdue Research Foundation Method of treatment using ligand-immunogen conjugates
EP2460889A3 (fr) * 2002-10-11 2012-07-04 Erasmus Universiteit Rotterdam Amorces d'acides nucléiques pour les'études de la clonalité des translocations BCL2-IGH basées sur PCR

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Publication number Priority date Publication date Assignee Title
JP7279633B2 (ja) * 2017-03-29 2023-05-23 東洋紡株式会社 核酸の検出方法

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EP0189628A1 (fr) * 1984-11-15 1986-08-06 The Wistar Institute Détection de néoplasme des B-cellules
WO1989008717A1 (fr) * 1988-03-14 1989-09-21 Board Of Regents, The University Of Texas System Detection des nombres minimaux de cellules neoplastiques portant des translocations d'adn par amplification des sequences d'adn
WO1989010979A1 (fr) * 1988-05-10 1989-11-16 E.I. Du Pont De Nemours And Company Procede de detection rapide d'acide nucleique

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EP0189628A1 (fr) * 1984-11-15 1986-08-06 The Wistar Institute Détection de néoplasme des B-cellules
WO1989008717A1 (fr) * 1988-03-14 1989-09-21 Board Of Regents, The University Of Texas System Detection des nombres minimaux de cellules neoplastiques portant des translocations d'adn par amplification des sequences d'adn
WO1989010979A1 (fr) * 1988-05-10 1989-11-16 E.I. Du Pont De Nemours And Company Procede de detection rapide d'acide nucleique

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NUCLEIC ACIDS RESEARCH. vol. 17, no. 5, 1989, ARLINGTON, VIRGINIA US page 2142; B.K.DE ET AL.: 'Multiple primer pairs for the detection of HTLV-I by PCR' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 85, July 1988, WASHINGTON US pages 4869 - 4873; M.CRESCENZI ET AL.: 'Thermostable DNA polymerase chain amplification of t(14;18) chromosome breakpoints and detection of minimal residual disease' cited in the application *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 87, no. 22, November 1990, WASHINGTON US pages 8923 - 8927; D.A.NICKERSON ET AL.: 'Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay' *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5595869A (en) * 1986-07-09 1997-01-21 The Wistar Institute Diagnostic methods for detecting lymphomas in humans
WO1995011990A1 (fr) * 1993-10-29 1995-05-04 Raggio-Italgene S.P.A. Dosage destine a la detection d'anomalies genetiques
US5985598A (en) * 1994-10-27 1999-11-16 Thomas Jefferson University TCL-1 gene and protein and related methods and compositions
US7749715B2 (en) 1994-10-27 2010-07-06 Thomas Jefferson University TCL-1 gene and protein and related methods and compositions
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IT1244983B (it) 1994-09-13
AU666648B2 (en) 1996-02-22
CA2109518A1 (fr) 1992-10-30
ITRM910286A1 (it) 1992-10-29
AU1649992A (en) 1992-12-21
JPH06509464A (ja) 1994-10-27
EP0583284A1 (fr) 1994-02-23

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