EP0991780A2 - Method and test kit for detecting malignant tumours - Google Patents
Method and test kit for detecting malignant tumoursInfo
- Publication number
- EP0991780A2 EP0991780A2 EP98942468A EP98942468A EP0991780A2 EP 0991780 A2 EP0991780 A2 EP 0991780A2 EP 98942468 A EP98942468 A EP 98942468A EP 98942468 A EP98942468 A EP 98942468A EP 0991780 A2 EP0991780 A2 EP 0991780A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- dna
- bax
- detection
- primer
- test kit
- 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.)
- Withdrawn
Links
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
- C12Q1/6876—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
- the invention relates to a method for the detection of malignant tumors on the basis of the detection of mutations in the Bax gene and to a test kit.
- Apoptosis or programmed cell death plays a crucial, physiological role in the regulation of cells and tissues.
- Dysregulations in this finely balanced system of activators and inhibitors of apoptosis play a decisive role in the development of malignant tumors.
- inactivators of apoptosis are overregulated and activators of apoptosis are inactivated and / or mutated or deleted. It follows that the exact, molecular biological analysis of the damage to the molecules involved provides important information about the pathogenesis of malignant diseases. In addition, the detection of damage in the relevant genes makes it possible to identify those that should be substituted in the context of gene therapy in order to enable causal therapy.
- a key activator of apoptosis mediation is represented by the Bax protein.
- This molecule belongs to the family of bcl-2 related genes that regulate the apoptosis ability of a cell.
- the protein bcl-2 is one of the inhibitors of apoptosis
- Bax is assigned to the genes that promote apoptosis.
- the Bax mRNA is expressed in three different splice variants, Bax-alpha, Bax-beta and two Bax-gamma forms. Bax-alpha is of particular importance for the induction of programmed cell death.
- the Bax gene it has recently been possible to show that specific damage to the DNA sequence is present in tumors of the digestive tract ("frame shift mutation”) / somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype; Rampino-H et al., (1997) Science 275 (5302): 967-9 /.
- the detection of this DNA damage is essential not only for the explanation of the development of tumors. Therefore, the Bax gene is also a candidate gene for the molecular diagnosis of malignant tumors, which should be replaced as part of gene therapy strategies in order to restore the tumor cell to programmed cell death.
- the invention was therefore based on the object of developing a clinically usable detection method for molecular characterization of mutative changes in the Bax gene, which avoids the disadvantages of radioactive methods and can be used for routine diagnostics.
- nucleic acids such as RNA and / or DNA
- the detection of malignant tumors is characterized in that the genomic DNA or the RNA is isolated from clinical sample materials and insertion and deletion mutations in the human Bax gene in a guanosine repeat tract of the nucleotide region within the coding region of the gene in exon-3 - Examine the range of nucleotides with guanosine bases by amplifying this DNA section using a primer pair, the primer pair being selected so that the amplification length is between 90 and 120 base pairs and a primer is labeled with a fluorescent dye.
- the amplified DNA fragment is applied to a sequencing gel or a separation matrix, separated and the insertions and deletions are detected over the length of the fragment.
- genomic DNA is isolated from the clinical tissue samples to be examined by treating the sample with a lysis binding buffer, e.g. Incubated guanidine isothiocyanate, lithium chloride or guanidine hydrochloride, optionally in combination with detergents and alcohols, as well as with a mineral carrier material for binding the genomic DNA.
- a lysis binding buffer e.g. Incubated guanidine isothiocyanate, lithium chloride or guanidine hydrochloride, optionally in combination with detergents and alcohols, as well as with a mineral carrier material for binding the genomic DNA.
- known silica materials ⁇ 50 nm are preferably used, particularly preferably non-porous silicon dioxide with a particle size of 7 nm-1 ⁇ m.
- the genomic DNA fixed to the carrier material under lysis binding buffer is applied to a membrane made of polysulfone ether with a pore size of 0.05 to 1 ⁇ m, preferably 0.2 ⁇ m to 0.5 ⁇ m.
- the carrier material with the genomic DNA is then fixed on the membrane by a centrifugation step or a vacuum filtration, washed on the membrane by adding a washing buffer of ethanol, sodium chloride and Tris-HCl and eluted with a low salt buffer of Tris-HCl and EDTA.
- the guanosine repeat tract to be examined for "frame shift" mutations within the coding region of the Bax gene is amplified enzymatically with a pair of primers, one of the primers being provided with a fluorescent label. After the enzymatic duplication, the duplicated DNA fragment is thus also provided with the fluorescent label.
- the primer pair is selected so that the amplification length is between 90 and 120 base pairs. This subsequently enables a highly sensitive electrophoretic separation of the amplified DNA fragment.
- a pair of primers of the sequence is preferred:
- Primer 1 5'-TCATCCAGGATCGAGCAGG-3
- Primer 2 5'-CTCGCTCAGCTTCTTGGTGG-3 used.
- the DNA fragment generated via the amplification reaction is separated for analysis of its exact length on a sequencing gel (e.g. ABI 373) or in a capillary with a separation matrix (e.g. ABI 310) and analyzed by means of the DNA sequencer.
- a sequencing gel e.g. ABI 373
- a separation matrix e.g. ABI 310
- the inventive method ideally solves the listed problems of radioactive techniques and allows simple and quick analysis of the Bax gene and thus a molecular characterization of malignant tumors.
- the invention is furthermore also suitable for subjecting non-invasively obtainable sample materials to a mutation test in the Bax gene, which means that early detection of malignant changes is also possible by detecting mutations in the Bax gene.
- the method according to the invention is particularly suitable for early detection or molecular characterization of tumors of the lungs, gastrointestinal tumors, preferably Lac, pancreas, and tumors of the hematopoietic system.
- non-invasively obtainable clinical sample materials e.g. Stool samples, lavage or exhaled condensates are fed to the mutation analysis. It has been shown that Bax frameshift mutations are not limited to gastrointestinal tumors.
- the method is also advantageously suitable for the examination of blood, saliva, swab materials or plasma samples. This is crucial for the possible early detection of malignant changes and for the planning of necessary therapeutic measures.
- the invention also relates to a test kit for the examination of clinical samples for mutations in the Bax gene, which contains all the necessary reagents for the examination of mutations in the Bax gene.
- a test kit for the examination of clinical samples for mutations in the Bax gene, which contains all the necessary reagents for the examination of mutations in the Bax gene.
- DNA extraction from a wide variety of sample materials and the generation of a fluorescence-labeled DNA fragment can be achieved.
- such a test kit also contains the necessary positive and negative controls and a fragment length standard.
- the test kit according to the invention is characterized by: 1. a DNA and / or RNA extraction system 2. primer mix including a fluorescence-labeled primer (e.g. a hex-labeled primer)
- Fragment length standard e.g. Rox-marked
- Such a test kit thus also allows people who are not highly specialized in molecular biology to carry out Bax gene diagnostics in a routine laboratory operation.
- the method on which the invention is based and the means for carrying out this method make it possible to routinely detect mutations in the Bax gene.
- the procedure is particularly advantageous if the number of tumor biopsy materials is very limited. Due to the higher number of copies of Bax-RNA molecules, the detection can be carried out very efficiently.
- the total RNA of the sample is isolated in a manner known per se.
- the isolated RNA is then rewritten into cDNA using reverse transcription and can then be examined again for the detection of frameshift mutations.
- the cell lines Lovo which has an insertion and a deletion mutation in the Bax guanosine extract (G) ⁇ , and the cell line SW620 (Bax wild type) were used.
- the DNA isolation of the genomic DNA was carried out from the cell lines as well as from cell line mixtures.
- the isolated genomic DNA was then used for the selective duplication of the DNA section of the Bax gene to be examined.
- DNA 1-5 ⁇ l DNA is used for the amplification reaction.
- a primer is used which is provided with a standard fluorescent label (hex label).
- 0.5 ⁇ l of the DNA fragment generated is mixed with 2 ⁇ l water and 2 ⁇ l formamide and incubated for 5 minutes at 95 ° C.
- This approach is applied to a 6% denaturing polyacrylamide sequencing gel and separated on an automatic sequencer (ABI 373) at 2500 V, 25 mA and 30 watts for 2 hours.
- the analysis result is shown in Figure 1. The percentages of LoVo or SW620 are given in brackets.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19727932 | 1997-07-01 | ||
DE19727932A DE19727932A1 (en) | 1997-07-01 | 1997-07-01 | Method and test kit for the detection of malignant tumors |
PCT/DE1998/001806 WO1999001573A2 (en) | 1997-07-01 | 1998-07-01 | Method and test kit for detecting malignant tumours |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0991780A2 true EP0991780A2 (en) | 2000-04-12 |
Family
ID=7834219
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98942468A Withdrawn EP0991780A2 (en) | 1997-07-01 | 1998-07-01 | Method and test kit for detecting malignant tumours |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0991780A2 (en) |
AU (1) | AU9061098A (en) |
DE (1) | DE19727932A1 (en) |
WO (1) | WO1999001573A2 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2615143C2 (en) | 2010-03-24 | 2017-04-04 | Адвирна | Self-delivered rnai compounds of reduced size |
WO2015084897A2 (en) | 2013-12-02 | 2015-06-11 | Mirimmune, Llc | Immunotherapy of cancer |
WO2015168108A2 (en) | 2014-04-28 | 2015-11-05 | Rxi Pharmaceuticals Corporation | Methods for treating cancer using nucleic targeting mdm2 or mycn |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19736715A1 (en) * | 1996-08-17 | 1998-02-19 | Invitek Gmbh | Reagent for tumour diagnosis |
-
1997
- 1997-07-01 DE DE19727932A patent/DE19727932A1/en not_active Ceased
-
1998
- 1998-07-01 EP EP98942468A patent/EP0991780A2/en not_active Withdrawn
- 1998-07-01 WO PCT/DE1998/001806 patent/WO1999001573A2/en not_active Application Discontinuation
- 1998-07-01 AU AU90610/98A patent/AU9061098A/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO9901573A2 * |
Also Published As
Publication number | Publication date |
---|---|
WO1999001573A2 (en) | 1999-01-14 |
AU9061098A (en) | 1999-01-25 |
WO1999001573A3 (en) | 1999-04-01 |
DE19727932A1 (en) | 1999-01-07 |
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Legal Events
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Inventor name: BENDZKO, PETER Inventor name: WOLFF, GERHARD Inventor name: DOERKEN, BERND Inventor name: HILLEBRAND, TIMO |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: INVITEK GESELLSCHAFT FUER BIOTECHNIK & BIODESIGN M |
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