EP1309728A2 - Procede d'analyse hautement parallele de polymorphismes - Google Patents

Procede d'analyse hautement parallele de polymorphismes

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Publication number
EP1309728A2
EP1309728A2 EP01942981A EP01942981A EP1309728A2 EP 1309728 A2 EP1309728 A2 EP 1309728A2 EP 01942981 A EP01942981 A EP 01942981A EP 01942981 A EP01942981 A EP 01942981A EP 1309728 A2 EP1309728 A2 EP 1309728A2
Authority
EP
European Patent Office
Prior art keywords
highly parallel
polymorphisms
probes
parallel characterization
allele
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Withdrawn
Application number
EP01942981A
Other languages
German (de)
English (en)
Inventor
Kurt Berlin
Ivo Glynne Gut
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.)
Epigenomics AG
Original Assignee
Epigenomics AG
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 Epigenomics AG filed Critical Epigenomics AG
Publication of EP1309728A2 publication Critical patent/EP1309728A2/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Withdrawn legal-status Critical Current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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention describes a high throughput analysis method for the parallel characterization of polymorphisms, in particular SNPs.
  • the method for analyzing DNA methylation can be used simultaneously or in a separate experiment.
  • the human genome project the first sequencing of the human genome, will be completed in the next few years. This project will make it possible to identify all of the approximately 100,000 genes.
  • the sequence information opens up unimagined possibilities for the elucidation of gene functions. This in turn opens up the possibility of doing pharmacogenetics and pharmacogenomics.
  • Pharmacogenetics and pharmacogenomics target the use of drugs depending on a genotype. The aim is to increase the effectiveness of medication.
  • the necessary intermediate step is to determine the polymorphisms and genotypes associated with a particular response. Therefore, increasingly efficient genotyping methods are required.
  • Microsatellites are highly polymorphic, ie they have a large number of alleles. They are characterized in that a repetitive sequence element with a different number of repetitions for different alleles is flanked by conserved sequences. There is an average of one microsatellite marker per million bases. A map of 5,000 positioned microsatellite markers has been published by CEPH (Dil. C., et al. Nature, March 14, 1994). Microsatellites are through ⁇ ⁇ rO (V)
  • CD d ⁇ Q rr I- 1 li O 0 N 0 s: 0 ⁇ - O ⁇ J d rt o ⁇ ? Hi 0 li ⁇ HS 0 ⁇ ⁇ 0 3 3 s: co ⁇ -. 0 0 li rt ffi Hl ⁇ o ⁇ - ⁇ - H iQ o-, C ⁇ JN PJ tc 0 ⁇ ⁇ - PJ O PJ: ⁇ • ⁇ d: ⁇ do co ⁇ J
  • oligonucleotide covers the sequence from the 5 'side right up to the SNP, so that the SNP joins the 3' end of this oligonucleotide.
  • a structurally active endonuclease removes the 5 'overhang from the fully complementary oligonucleotide.
  • the trimmed overhang is analyzed using mass spectrometry and used to identify the allele.
  • a disadvantage of the method shown is that the products have to be cleaned thoroughly before the mass spectrometric analysis. Magnetic beads that are not easy to use are used for this purification. This is a major disadvantage of many genotyping methods that use mass spectrometry for analysis.
  • Another genotyping method is the Taq Man Assay. In this allele-specific enzyme-specific separation of a fluorescence quencher from a fluorescence dye-bearing 0-ligonucleotide is carried out.
  • MALDI Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
  • MALDI has revolutionized the analysis of biomolecules (Karas, M. & Hillenkamp, F. Anal. Chem. 60, 2299-2301 (1988)).
  • MALDI has been used in various variants for the analysis of DNA. The variants range from primer extension to sequencing (Liu, Y.-H., et al. Rapid Commun. Mass Spectrom. 9, 735-743 (1995); Ch'ang, L.-Y., et al. Rapid Commun Mass Spectrom. 9, 772-774 (1995); Little, DP, et al. J. Mol, Med. 75, 745-750 (1997); Haff, L. & Smirnov, IP Genome Res. 7, 378- 388 (1997), Fei, Z., Ono, T. & Smith, LM
  • the object of the present invention is to provide a method for highly parallel genotyping of polymorphisms.
  • the problem is solved by a method for the highly parallel characterization of polymorphisms, whereby one carries out the following steps: a) a set of probes, which is provided with at least one detectable marking which is characteristic of the respective probe, is bound to an addressed surface, the binding of the probes generated to the surface being cleavable again photochemically, chemically or enzymatically ; b) a nucleic acid to be investigated is hybridized to these probes; c) the probes are changed in an allele-specific enzymatic reaction; d) a part of the probes is removed which is meaningless for the analysis of the allele-specific reaction; e) the allele-specific products are analyzed on the basis of the detectable markings and the alleles present in the nucleic acid sample queried are determined.
  • the address of the surface in step a) is the position (oligonucleotide array), a color, a fluorescent label, an isotopic label, a chemical label or a radioactive label.
  • the probes are oligonucleotides, modified oligonucleotides, peptide nucleic acids (PNAs), chimera of these classes of compounds or other substances which interact with DNA in a sequence-specific manner.
  • PNAs peptide nucleic acids
  • the probes are bound to the surface via reversible binding systems.
  • the nucleic acids to be examined in step b) are genomic DNA, nated DNA, cDNA, RNA, PCR products or ligation products.
  • the probes are converted into specific products according to step c) depending on the respective sequence of the template hybridized thereon by means of a polymerase and nucleotide building blocks.
  • sequencing is carried out by simultaneous use of deoxy and dideoxynucleotide building blocks and not only polymorphisms are detected.
  • the probes are converted into specific products in accordance with step c) depending on the particular sequence of the template hybridized thereon by means of a ligase and a phosphorylated oligonucleotide.
  • the probes are cut in an all-specific manner as a function of the respective sequence of the template hybridized thereon by means of a helper oligonucleotide and a structurally active endonuclease. It is also preferred that the allele-specific products are analyzed by means of mass spectrometry.
  • MALDI matrix-assisted laser desorption / ionization mass spectrometry
  • electrospray ionization mass spectrometry is used for the analysis.
  • the allele-specific products are present in a manner that is particularly well suited for mass spectrometric analysis. It is further preferred according to the invention that the allele-specific products according to step d) are shortened using an enzymatic or chemical method. It is particularly preferred that the particularly good suitability for mass spectrometric analysis arises from the fact that the allele-specific products have a net simply positive or simply negative charge. It is further preferred that a chemical reaction is used to neutralize charges that would otherwise contribute to a neutral or multiple negative net charge of the product. Thus, it is also preferred that the phosphate groups, thiophosphate groups or dithiophosphate groups of the oligonucleotide backbone are charge neutralized by a selective alkylation reaction. It is also particularly preferred according to the invention that the simple charge is achieved according to the invention by introducing a chemical function which carries the charge.
  • the reversible binding chemistry contributes the simple charge to the product by means of an induced break. It is preferred that the induced bond break occurs chemically or photochemically. It is further preferred that the induced bond break occurs during a desorption process of the analysis process.
  • a matrix is applied to the surface which supports the desorption in the MALDI process. It is again preferred that the induced bond break is induced by the matrix.
  • a multiplicity of are different probes. It is further preferred that allele-specific products of the probes result in an unambiguous mass as a detectable label in the analysis after the allele-specific reaction after step c) after cleavage from the surface. In addition, it is preferred that allele-specific products of the probes result in an unambiguous pattern of fragment masses as a detectable label by the allele-specific reaction after step c) after cleavage from the surface in the analysis. It is particularly preferred here that the masses of all products or product fragments of the allele-specific reactions allow a clear conclusion to be drawn about the alleles present in the nucleic acid queried.
  • known polymorphisms are genotyped in the DNA to be examined.
  • unknown polymorphisms are identified in the DNA to be examined.
  • cytosine methylations are detected and visualized.
  • the chemical treatment of the DNA is carried out with a bisulfite solution (disulfite, hydrogen sulfite).
  • the amplification is carried out by means of the polymerase reaction (PCR).
  • the invention thus describes a method for the highly parallel characterization of polymorphisms.
  • a set of probes are bound to an addressed surface.
  • the probes used are preferably oligonucleotides, modified oligonucleotides, peptide nucleic acids (PNAs), chimera of these classes of compounds or other substances which interact with DNA in a sequence-specific manner.
  • PNAs peptide nucleic acids
  • the respective probe is provided with a characteristic, detectable marking.
  • This marking is particularly preferably the mass of a fragment of the probe.
  • the addressing of the surface is the position (oligonucleotide array), a color, a fluorescent label, an isotopic label, a chemical label or a radioactive label.
  • the generated binding of the probes to the surface can be cleaved again photochemically, chemically or enzymatically.
  • the probes are bound to the surface via reversible binding systems.
  • the nucleic acid to be investigated which is preferably composed of genomic DNA, cloned DNA, pretreated DNA, cDNA, RNA, is hybridized. PCR products or ligation products exist on said probes.
  • the DNA is preferably treated beforehand with a bisulfite solution (disulfite, hydrogen sulfite).
  • the probes are changed in an allele-specific enzymatic reaction.
  • the probes are converted into specific products by means of a polymerase and nucleotide building blocks, depending on the particular sequence of the hybridized template.
  • the probes are converted into specific products by means of a ligase and a phosphorylated oligonucleotide, depending on the particular sequence of the hybridized template.
  • the probes are then cut allele-specifically, depending on the particular sequence of the template hybridized thereon, with a helper oligonucleotide and a structurally active endonuclease.
  • methylation patterns in the pretreated DNA to be analyzed can be examined.
  • SNPs are examined in the pretreated DNA to be analyzed.
  • a large number of different probes are preferably located on an addressed analysis point on the surface.
  • the allele-specific products are preferably shortened using an enzymatic or chemical method.
  • the allele-specific products are analyzed on the basis of the detectable markings and the determination of the alleles present in the queried nucleic acid sample is carried out.
  • the allele-specific products are analyzed by means of mass spectrometry.
  • the allele-specific products are preferably in a type which is particularly suitable for mass spectrometric analysis.
  • the particularly good suitability for mass spectrometric analysis preferably arises from the fact that the allele-specific products are net positively or simply negatively charged.
  • a chemical reaction is used to neutralize charges, which would otherwise contribute to a neutral or multiple negative net charge of the product.
  • phosphate groups, thiophosphate groups or dithi Charge neutralized ophosphate groups of the oligonucleotide backbone by a selective alkylation reaction.
  • the simple charge comes about by introducing a chemical function that carries the charge.
  • the reversible binding chemistry preferably contributes the simple charge to the product through an induced break.
  • the induced bond break occurs chemically or photochemically.
  • the induced bond break takes place during the desorption process of the analysis process.
  • a matrix is preferably applied to the surface that supports desorption in the MALDI process.
  • the induced breaking of the bond is induced by the matrix.
  • matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or electron spray ionization mass spectrometry are used for the analysis.
  • the extension products of the probes result in an unambiguous mass as detectable marking in the analysis due to the allele-specific reaction after being split off from the surface.
  • Known polymorphisms can thus preferably be identified in the DNA to be examined.
  • the extension products of the probes result from the allele-specific reaction after cleavage from the surface in the analysis, a clear pattern of fragment masses as a detectable label. Unknown polymorphisms can thus preferably be identified in the DNA to be examined.
  • Fig. La and lb an illustration of the process steps using an example
  • Fig. 2 shows a possible immobilization of the probes on the 0- surface comprising a photolabile linker.
  • FIGS. 1 a and 1 b The following steps are shown in FIGS. 1 a and 1 b:
  • the nucleic acid to be investigated is then hybridized to the probe.
  • the probes are then extended in an allele-specific reaction. 4.
  • the nucleic acid to be examined is removed. 5. Subsequently, a part of the probes is removed which is meaningless for the allele-specific reaction.
  • a mass spectrometer is preferably used for the analysis, which identifies the elongated probes on the basis of their masses and which at the same time enables detachment from the surface (eg photolytic reaction in a laser desorption mass spectrometer).
  • FIG. 2 shows a possible linkage of the primers to the surface.
  • the nucleotide shown is part of the primer, which is not shown for the sake of clarity.
  • the genomic DNA sample to be analyzed is first subjected to a bisulfite reaction in which, as is known to the person skilled in the art, non-methylated cytosine residues are converted into uracil residues.
  • the fragment of the bisulfited DNA to be analyzed is then amplified by a polymerase chain reaction using specific primers.
  • a template-directed extension of the primer TCTATTTACTTCATTCCACTTAAsT * sC is catalyzed by thermosequenase.
  • the bisulfite-treated and amplified by means of a PCR reaction serves as a template for the primer extension
  • the primer has two phosphothioate bonds which are particularly marked by "s".
  • the base of the penultimate monomer here particularly marked by "* ⁇ ", is modified with a substituent bearing a quaternary amino function.
  • the primer extension is carried out in the presence a mixture of phosphothioate-modified dideoxynucleotides, the incorporation of which leads to the formation of a third phosphothioate bond at the 3 'end of the primer extended by one nucleotide block.
  • a mixture of ⁇ -sddATP and ⁇ -sddGTP is used, so that this corresponds to the methylation status of the template on which the underlying genomic DNA sample is based
  • a mixture of the extension products TCTATTTACTTCATTCCACTTAAsT * sCsA and TCTATTTACTT-CATTCCACTTAAsT * sCsG which do not have a 3 'hydroxyl group, is initiated by a three-minute denaturation step at 95 ° C and then analogous to a PCR reaction forty cycles performed.
  • the pH is lowered to below 7.0 by adding acetic acid.
  • the reaction solution is incubated at 37 ° C for 45 minutes. During this time, the DNA Teplat is completely digested, while the extended primer is only incompletely digested while maintaining the phosphothioate bonds to form the hydrolysis products AsT * sCsA and AsT * sCsG.
  • Example ⁇ -cyano-4-hydroxycinnamic acid methyl ester mixed, and applied to a MALDI target.
  • the measurement was carried out on a Bruker Reflex II TOF mass spectrometer in positive ion mode.
  • FIG. 3 shows spectra which can be assigned to different degrees of methylation of the investigated methylation site in the genomic DNA sample.
  • Figures 3-5 show MALDI-TOF spectra obtained after analyzing the methylation status of the first methylation site of exon 14 of the factor VIII gene in accordance with Example 1:
  • Fig. 3 The methylation position was completely methylated in the genomic DNA (primer extension by ⁇ -sddGTP)
  • Fig. 4 Methylation position was partially methylated in the genomic DNA (primer extension by both ⁇ -sddGTP and ⁇ -sddATP)
  • the example describes the execution of the assay set out in the first example in slide format.
  • the surface of slides offers the possibility of completely removing excess reagents after each individual step by means of suitable washing processes.
  • Untreated slides are washed and chemically treated with an aminosilane.
  • one or more spacers such as aminoethoxyethoxyacetic acid (AEEA) can be coupled using suitable chemical methods.
  • the terminal amino group is then provided with a photochemically cleavable linker group, preferably 4- [4- (1- (Fmocamino) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid.
  • a homobifunctional reagent such as, for example, ethylene glycol bis-succinimidyl succinate (Pierce) in accordance with the manufacturer's instructions.
  • Various amino-modified primers such as TCTATTTACTTCATTCCACTTAAsT ⁇ C can now be covalently immobilized on the surface obtained.
  • TCTATTTACTTCATTCCACTTAAsT ⁇ C can now be covalently immobilized on the surface obtained.
  • the primer is extended on the slide surface in analogy to the 1st example, forming a surface-bound mixture of the extension products TCTATT- TACTTCATTCCACTTAAsT * CsA and TCTATTTACTTCATTCCACT- TAAsT ff CsG.
  • the 5 '-phosphorodiesterase digestion and the subsequent methylation of the phophhothioate bonds remaining in the degradation products are also carried out analogously to the first example, with the entire slide or a defined section of its surface being brought into contact with the corresponding reagents. After the methylation has taken place, the slide is washed with a suitable solvent before the photoliker is cleaved by irradiation with UV light of a suitable wavelength.
  • the resulting cleavage products contain two positions from the 3 'end which removes a phosphodiester bond which is not methylated under the alkylation conditions and whose negative charge provides highly sensitive detection of the reaction products
  • MALDI-TOF enables.
  • the MALDI measurement is carried out in negative ion mode directly on the slide surface.
  • a matrix solution such as hydroxypicolinic acid or trihydroxyacetophenone
  • the measured masses of the fragments resulting from the primer extension products in conjunction with their relative intensity, allow the methylation status of the investigated methylation position in the original DNA to be determined in analogy to the first example.
  • This example describes another way to
  • the methylation of the phosphothioate bonds in the primer extension products is carried out directly after the extension reaction Digestion is carried out in a position-specific manner by precisely applying a droplet of enzyme solution to a freely selectable position on the slide surface and then incubating at 100 percent atmospheric humidity.
  • the nuclease digestion results in the detachment of the alkylated P on the one hand Rimer extension products from the slide surface and on the other hand in the formation of the same products as under Example 1, which can be detected with high sensitivity by MALDI-TOF measurement in positive ion mode.

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Abstract

La présente invention concerne un procédé permettant la caractérisation hautement parallèle de polymorphismes, en particulier de polymorphismes à simple nucléotide (SNP), ledit procédé pouvant également servir à la détection simultanée ou séparée de méthylations d'ADN. Pour commencer, un ensemble d'échantillons qui est pourvu d'au moins une marque identifiable caractéristique de l'échantillon correspondant, est lié à une surface adressée, la liaison faite entre l'échantillon et la surface pouvant être à nouveau rompue par voie photochimique, chimique ou enzymatique. Pour finir, un acide nucléique à étudier est hybridé au contact de ces échantillons, les échantillons sont modifiés dans une réaction enzymatique allèle spécifique, et une partie des échantillons qui s'avère inutile à l'analyse de la réaction allèle spécifique, est éliminée. Les produits allèle spécifiques sont tout d'abord analysés grâce aux marques identifiables et une détermination des allèles présents dans l'échantillon d'acide nucléique analysé est faite.
EP01942981A 2000-04-25 2001-04-25 Procede d'analyse hautement parallele de polymorphismes Withdrawn EP1309728A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10021204 2000-04-25
DE10021204A DE10021204A1 (de) 2000-04-25 2000-04-25 Verfahren zur hochparallelen Analyse von Polymorphismen
PCT/DE2001/001607 WO2001081620A2 (fr) 2000-04-25 2001-04-25 Procede d'analyse hautement parallele de polymorphismes

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Cited By (2)

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US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US12435375B2 (en) 2018-04-02 2025-10-07 Grail, Inc. Methylation markers and targeted methylation probe panel

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DE202006020290U1 (de) * 2006-03-13 2008-02-28 Schubert, Adrian Vorrichtung zur Erkennung und Identifizierung von Zielstrukturen

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
US12435375B2 (en) 2018-04-02 2025-10-07 Grail, Inc. Methylation markers and targeted methylation probe panel
US11410750B2 (en) 2018-09-27 2022-08-09 Grail, Llc Methylation markers and targeted methylation probe panel
US11685958B2 (en) 2018-09-27 2023-06-27 Grail, Llc Methylation markers and targeted methylation probe panel
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US12410482B2 (en) 2018-09-27 2025-09-09 Grail, Inc. Methylation markers and targeted methylation probe panel

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WO2001081620A3 (fr) 2003-02-13
DE10191554D2 (de) 2003-06-12
WO2001081620A2 (fr) 2001-11-01
DE10021204A1 (de) 2001-11-08
AU2001265759A1 (en) 2001-11-07

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