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WO1996000796A1 - Procede de detection de mutations - Google Patents

Procede de detection de mutations Download PDF

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Publication number
WO1996000796A1
WO1996000796A1 PCT/NL1995/000227 NL9500227W WO9600796A1 WO 1996000796 A1 WO1996000796 A1 WO 1996000796A1 NL 9500227 W NL9500227 W NL 9500227W WO 9600796 A1 WO9600796 A1 WO 9600796A1
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WO
WIPO (PCT)
Prior art keywords
fragment
label
pattern
light
gel
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.)
Ceased
Application number
PCT/NL1995/000227
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English (en)
Inventor
Erik Mullaart
Jan Vijg
Gerrit Johannis De Vos
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.)
Ingeny BV
Original Assignee
Ingeny BV
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ingeny BV filed Critical Ingeny BV
Priority to AU26842/95A priority Critical patent/AU2684295A/en
Publication of WO1996000796A1 publication Critical patent/WO1996000796A1/fr
Anticipated expiration legal-status Critical
Ceased 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
    • C12Q1/6813—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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a method for detecting mutations in the genome of an individual.
  • the genetic material differs from individual to individual because each genome is unique. Genetic variation can be determined by means of hybridization analysis after electrophoretic separation of DNA fragments which are generated by fragmenting for instance the genomic DNA.
  • the electrophoretic pattern can be transferred from the gel to a nitrocellulose filter.
  • a radioactively labelled probe the parts of the genome which hybridize with the probe can be made visible using an X-ray photo of the filter.
  • the position of the visualized fragment is a measure of the size of the fragment. Variation between individuals in the position of a band when one probe is used indicates polymorphism. This is caused by mutations in particular regions in the genome. Such mutations may be base substitutions but also insertions and deletions.
  • the running behaviour of a fragment in a gel is influenced by such mutations.
  • Polymorphism can for instance be used to identify an individual such as in determining paternity and in forensic medicine.
  • a high degree of polymorphism is generally encountered in non-coding parts of the genome. Logically, less polymorphism occurs in the genes. Genes can however be slightly mutated. Such mutations can result in mutations in the coded proteins or even in total deactivation of the gene. Such phenomena can cause particular clinical pictures or other disorders.
  • fragments appear in the form of a dark spot on the film.
  • the position of a fragment is specific to its length and base pair composition. However, no distinction can be made between fragments which appear at the same position. With the known methods only one set of fragments can thus be analysed at any one time.
  • the known method is very labour- intensive due to the different steps.
  • a separate gel and a separate filter as well as an X-ray film are required for each set of DNA fragments for testing.
  • These materials, and also the chemicals required to develop the film of course also entail additional costs.
  • Waste chemicals also form an impact on the environment and radioactive probes can damage the health of those operating them. Work with radioactive probes moreover results in radioactive waste.
  • the result of the experiment can only be obtained after some time.
  • the advantage of the method according to the invention is that a number of persons or a number of genes can be scanned on a single polyacrylamide gel. This saves much time as well as material
  • the separation is preferably a two-dimensional separation, wherein the mixture is separated in one direction on the basis of differences in size and in the other direction on the basis of differences in base pair composition of the fragments.
  • the separation in the second dimension can for instance take place in a denaturing gradient. Fragments with a high GC content will melt and stop at a higher concentration of the denaturing agent than fragments with a high AT content. In this manner differences in base pair composition are made visible.
  • a two-dimensional separation thereby has a higher resolution than a one-dimensional separation.
  • the DNA fragments for testing are amplified using a DNA- amplification method. Such an amplification method is for instance the polymerase chain reaction (PCR) . With the amplified fragments a heteroduplexing is performed. The fragments of an individual are then provided with a label, for example a fluorochrome, specific to that individual. The fragments are thereafter separated in two directions on a polyacrylamide gel.
  • PCR polymerase chain reaction
  • the label is preferably a fluorochrome which, after being excited with visible or invisible light of a determined wavelength, returns while emitting light with a wavelength specific to that fluorochrome.
  • any other chemical compound can be used which can be linked to a DNA molecule, can be excited in one way or another and returns within a short time to the initial state while emitting light of a specific wavelength. Excitation can optionally also take place using electromagnetic radiation.
  • the labels can also be made visible independently of each other or in any desired combination. The patterns of a large number of patients could thus be compared one by one with the control. Further understood by. "unique label" is any other molecule which is distinguished from other corresponding molecules by means of a detectable characteristic.
  • fluorochromes can be envisaged elements which each have a unique NMR spectrum.
  • elements which can be used are gold, silver, nickel, iron etc.
  • the fragments of a person are then provided with an element unique to that person which can later be distinguished using for instance NMR, X-ray diffraction or other techniques.
  • the label is formed by fluorochromes which, after excitation, each emit light with a clearly discernible colour.
  • the invention further relates to a device for performing the method according to the invention.
  • the device consists of a substrate for a gel, a light source placed on one side of the gel which emits light of a wavelength range such that all the excitabfe TabeTs occurring in the label pattern can be excited therewith, a filter placed on the other side of the gel which allows through light emitted by the excitable labels but not the light emitted by the light source, and means for detecting the pattern of the light emitted by the excitable labels.
  • the label pattern can of course also be detected by eye. In the case of a small number of patients with clearly distinguishable colour labels this is still feasible. However, as the number of patients becomes larger and the labels less easy to distinguish from each other, the device preferably contains a scanner which can detect the wavelength emitted by each spot. In preference the device is further provided with a processing unit for analysing the light pattern detected by.the detecting means. When it is possible to standardize the separation the results can be compared automatically with data stored in the processor unit. Diagnostics can hereby be largely automated. When recording of the emitted label pattern is desired the device can also contain a recording unit. A photo or video camera can be envisaged here.
  • FIG. 3 shows a schematic view of an embodiment according to the invention.
  • the gel 1 is situated on a substrate (not shown)
  • the substrate can be a separate substrate but may also be formed by one or both glass plates of the original gel arrangement.
  • the gel may also be placed directly onto the filter.
  • the filter serves herein as substrate.
  • a filter 2 is situated on one side of the gel.
  • a light source 3 with a suitable wavelength range, for instance an UV lamp.
  • a scanner 4 On the side of the filter remote from the gel. Filter 2 does not allow the light emitted by the lamp 3 and falling through the gel through to the scanner 4.
  • the light emitted by the fluorochromes in the gel 1 is however allowed through. Using the scanner it can be determined which wavelength is being emitted at which position. On the basis of this data the spot pattern can be analysed.
  • the gene which, when mutated, results in the disease cystic fibrosis is compared with the corresponding non-mutated gene.
  • An amplification of the exons of the gene is carried out by means of PCR.
  • the thus formed fragments of the mutated gene, which substantially enclose the whole exons, are labelled with a yellow, light-emitting fluorochrome.
  • the fragments which are formed starting from the non-mutated gene are labelled with a blue, light-emitting fluorochrome.
  • Both sets of fragments are then mixed and separated on a two-dimensional polyacrylamide gel. After separation the gel is exposed, whereby the fluorochromes are excited. The yellow and blue light subsequently emitted by the fluorochromes is detected as a green spot at positions where the fragments are identical, as a blue spot at a position where a fragment of the non-mutated gene has appeared and as a yellow spot at positions where the mutated fragments of the mutated gene are present.
  • Figure 1A is a gel having only the fragments of one patient. The fragments of the non-mutated gene are made visible in the gel of figure IB.
  • Figure 1C shows the combination of both gels.
  • non-mutated gene is combined in the same, manner as in example 1 with two different mutated genes.
  • the non-mutated gene is again labelled with a blue colour, the mutated genes are labelled with respectively red and green fluorochromes.
  • the mixed colour of these three colours of light is white. After exposure white light is emitted at all positions where fragments of the three individuals are situated. The greater part of the fragments is not detectable.
  • a mutated fragment of the red-labelled person is visible as a red light spot. Only the combination of blue and green remains at the original position of that fragment because the red light has disappeared there. At the positions where the green light has disappeared there remains a purple spot, while the green spot is visible at another position.
  • the present invention provides a method and device with which a large number of individuals can be tested for mutations in their genome in rapid and simple manner.
  • the invention is of particular significance in diagnostics, but is not limited thereto.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé de détection de mutations dans le génome d'un individu, consistant à amplifier au moins un fragment à tester provenant du génome; à associer au(x) fragment(s) d'ADN un marqueur caractéristique de l'individu; à fournir un ou plusieurs fragments de référence munis d'un autre marqueur caractéristique et correspondant au(x) fragment(s) à tester; à mélanger le(s) fragment(s) au(x) fragment(s) de référence; puis à procéder à la décomposition du mélange par électrophorèse; à rendre visibles les marqueurs caractéristiques; et à comparer la configuration de marqueurs du (des) fragment(s) à tester avec celle du (des) fragment(s) de référence. Ce procédé permet de dépister les mutations dans les échantillons d'ADN de plusieurs individus en même temps.
PCT/NL1995/000227 1994-06-28 1995-06-28 Procede de detection de mutations Ceased WO1996000796A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU26842/95A AU2684295A (en) 1994-06-28 1995-06-28 Method for detecting mutations

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9401082 1994-06-28
NL9401082A NL9401082A (nl) 1994-06-28 1994-06-28 Werkwijze voor het detecteren van mutaties.

Publications (1)

Publication Number Publication Date
WO1996000796A1 true WO1996000796A1 (fr) 1996-01-11

Family

ID=19864381

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL1995/000227 Ceased WO1996000796A1 (fr) 1994-06-28 1995-06-28 Procede de detection de mutations

Country Status (3)

Country Link
AU (1) AU2684295A (fr)
NL (1) NL9401082A (fr)
WO (1) WO1996000796A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317239A2 (fr) * 1987-11-13 1989-05-24 Native Plants Incorporated Procédé et dispositif pour la détection des polymorphismes de restriction des longueurs de fragments
EP0349024A1 (fr) * 1988-05-02 1990-01-03 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Un procédé pour la détermination simultanée des variations de la séquence d'ADN, et un kit à cet effet
EP0364255A2 (fr) * 1988-10-12 1990-04-18 Baylor College Of Medicine Amplification d'ADN des génomes multiples pour détecter des délétions
WO1992013101A1 (fr) * 1991-01-25 1992-08-06 Ingeny B.V. Procede de detection de la variation de sequences d'adn
WO1995007361A1 (fr) * 1993-09-10 1995-03-16 Institut Pasteur Procede de detection de molecules contenant des mesappariements nucleotidiques et de localisation de ces mesappariements, et application a la detection de substitutions ou de deletions de bases dans des sequences nucleotidiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317239A2 (fr) * 1987-11-13 1989-05-24 Native Plants Incorporated Procédé et dispositif pour la détection des polymorphismes de restriction des longueurs de fragments
EP0349024A1 (fr) * 1988-05-02 1990-01-03 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Un procédé pour la détermination simultanée des variations de la séquence d'ADN, et un kit à cet effet
EP0364255A2 (fr) * 1988-10-12 1990-04-18 Baylor College Of Medicine Amplification d'ADN des génomes multiples pour détecter des délétions
WO1992013101A1 (fr) * 1991-01-25 1992-08-06 Ingeny B.V. Procede de detection de la variation de sequences d'adn
WO1995007361A1 (fr) * 1993-09-10 1995-03-16 Institut Pasteur Procede de detection de molecules contenant des mesappariements nucleotidiques et de localisation de ces mesappariements, et application a la detection de substitutions ou de deletions de bases dans des sequences nucleotidiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
IWAHANA ET EL.: "Multiple fluorescence-based PCR-SSCP anlysis", BIOTECHNIQUES, vol. 16, no. 2, NATICK, MA US, pages 296 - 305 *

Also Published As

Publication number Publication date
AU2684295A (en) 1996-01-25
NL9401082A (nl) 1996-02-01

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