EP1135526A2 - Procede d'identification de modeles de methylation de cytosine dans l'adn genomique - Google Patents

Procede d'identification de modeles de methylation de cytosine dans l'adn genomique

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Publication number
EP1135526A2
EP1135526A2 EP99962069A EP99962069A EP1135526A2 EP 1135526 A2 EP1135526 A2 EP 1135526A2 EP 99962069 A EP99962069 A EP 99962069A EP 99962069 A EP99962069 A EP 99962069A EP 1135526 A2 EP1135526 A2 EP 1135526A2
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Prior art keywords
dna
positions
genomic dna
individuals
methylcytosine
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German (de)
English (en)
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Kathrin Berlin
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Epigenomics AG
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Epigenomics AG
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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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 invention relates to a method for identifying 5-methylcytosine positions in genomic DNA.
  • the genetic information that is obtained by completely sequencing genomic DNA as a base sequence describes the genome of a cell only incompletely.
  • 5-Methylcytosine nucleobases which result from the reversible methylation of DNA in the cell, are an epigenetic information carrier and are used, for example, to regulate promoters.
  • the methylation state of a genome represents the current status of gene expression, similar to an mRNA expression pattern.
  • 5-Methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, genomic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is therefore of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. Unfortunately, in the case of PCR amplification, the epigenetic information carried by the 5-methylcytosines is completely lost, and there is no method for obtaining this information through an amplification step.
  • DD 293 139 A5 describes a method for characterizing certain DNA sequences in which the DNA molecules whose unmethylated recognition sites are to be cut by a corresponding restriction endonuclease are mixed with a second, unmethylated DNA species (especially Oligonucleotide duplexes containing the recognition site) incubated.
  • WO 97/46705 A1 discloses a method for the detection of a methylated nucleic acid containing CpG, wherein the sample containing nucleic acid is brought into contact with a reagent which modifies unmethylated cytosine, the nucleic acid containing CpG in the sample is amplified by means of CpG-specific oligonucleotide primers, the oligonucleotide primer between modified me- distinguishes thylated and unmethylated nucleic acids and detects the methylated nucleic acids.
  • US Pat. No. 5,824,471 A1 describes a method for determining deviations between two nucleic acid strands, a multiplicity of duplexes being formed from the two strands or parts thereof and this duplex being brought into contact with a first and a second different bacteriophage resolvase, and wherein it is then determined from which bacteriophage resolvase the duplex is cleaved, the differences being determined in this way.
  • Sequence information also has to be determined less and less because the genome projects whose goal is the complete sequence of different organisms is to progress rapidly. Although only about 5% of the human genome is currently fully sequenced, an additional 5% is added every year because other genome projects are coming to an end, which frees up sequencing resources. The sequencing of the human genome is expected to be completed by 2006.
  • Matrix-assisted laser desorption / ionization mass spectrometry is a new, very powerful development for the analysis of biomolecules (Karas, M. and Hillenkamp, F. 1988. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 60: 2299-2301).
  • An analyte molecule is embedded in a matrix that absorbs in the UV. The matrix is evaporated into a vacuum by a short laser pulse and the analyte is thus transported unfragmented into the gas phase.
  • An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, ions are accelerated to different extents. Smaller ions reach the detector earlier than larger ones.
  • the flight time is converted into the mass of the ions.
  • this technology is able to distinguish molecules with a mass difference of 1 Da in the mass range from 1,000 to 4,000 Da. Due to the natural distribution of isotopes, most biomolecules are already about 5 Da wide.
  • this mass spectrometric method is ideally suited for the analysis of biomolecules. Products to be analyzed that are to be differentiated must reasonably be at least 5 Da apart. In this mass range, 600 molecules could be distinguished. In the range between 4,000 and 100,000 Da, isotope resolution is no longer achieved, but this range is also applicable. bar.
  • IR infrared
  • the use of an infrared (IR) laser coupled with the MALDI analysis of DNA has recently been described (Berkenkamp, S., Kirpkar, F. and Hillenkamp, F. 1998. Infrared MALDI mass spectrometry of large nucleic acids. Science. 281: 260 -262). This combination made it possible to detect DNA fragments with a size of up
  • Chemical mismatch cleavage is a method with which small differences between two DNA single strands can be shown (Cotton, RGH, Rodriguez, NR and Campbell, RD 1988. Reactivity of cytosine and thy- mme in smgle-base-pair mismatches with hydroxylammes and osmium tetroxide and lts application to the study of mutations. Proc. Natl. Acad. Sei. USA. 85: 4397-4401; Cotton, RGH 1993. Current methods for mutat on detection. Mut. Res. 285: 125-144; Saleeba , JA and Cotton, RGH 1993. Chemical cleavage of mismatch to detect mutations. Methods in Enzymology.
  • Muts Another possibility to identify non-complementary base pairs in heteroduplex DNA is to use enzymes such as Muts, which bind to non-complementary base pairs' Smith, J. and Modrich, P. 1996. Mutation detection with MutH, MutL and MutS mismatch repa r protems. Proc. Natl. Acad. Be. USA 93: 4374-4379; Parsons, BL and Heflich, RH 1997. Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA. Courage. Res. 374: 277-285).
  • Muts which bind to non-complementary base pairs' Smith, J. and Modrich, P. 1996. Mutation detection with MutH, MutL and MutS mismatch repa r protems. Proc. Natl. Acad. Be. USA 93: 4374-4379; Parsons, BL and Heflich, RH 1997. Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA. Courage. Res. 374
  • the object of the present invention is therefore to provide a method for the inexpensive and parallelisable detection of epigenetic information carriers in the form of 5-methylcytosine bases in genomic DNA.
  • the object is achieved according to the invention by a method for identifying 5-methylcytosine positions in genomic DNA, the following method steps being carried out: a) the genomic DNA of a cell, a cell line, a tissue or an individual is chemically treated in such a way that cytosine and 5-Methylcytosine react differently and there is a different base pairing behavior of the two products in the duplex, b) the same nucleic acid section is amplified by means of a polymerase reaction, c) the same nucleic acid section is treated at least one further cell, cell line, tissue or individual or any one Reference DNA corresponding to steps a) and b), d) is formed from the at least two amplificates Steps b) and c) heteroduplex, e) a displayable marker is introduced into the heteroduplex by a reaction which is specific for non-complementary base pairs.
  • a disulfite bisulfite, pyrosulfite
  • step a a disulfite (bisulfite, pyrosulfite) is used in step a) as a reagent for the selective conversion of cytosine to uracil, 5-methylcytosine remaining unchanged.
  • genomic DNA from several individuals, tissues, cell lines or cells is amplified together in step b).
  • genomic DNA from several individuals, tissues, cell lines or cells is amplified separately and then treated together in accordance with step e).
  • step d) by forming heteroduplexes with a completely methylated reference DNA, base mismatches occur at the positions at which cytosine was found in the genomic DNA. It is also preferred that in step d), by forming heteroduplexes with a completely demethylated reference DNA, base mismatches occur at the positions at which 5-methylcytosine was found in the genomic DNA.
  • the base mismatches lead to a specific or sufficiently selective backbone cleavage at these positions by means of "chemical mismatch cleavage" (chemical change at non-complementary positions).
  • the DNA is cleaved enzymatically specifically or sufficiently selectively at the base mismatches.
  • 1 DNA fragments are obtained in step e), the size of which allows conclusions to be drawn about the cleavage positions and thus the position of the methylcytosines and / or the methylation positions which vary between different individuals, tissues, cell lines or cells allowed.
  • fragments are analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI).
  • MALDI matrix-assisted laser desorption / ionization time-of-flight mass spectrometry
  • the fragments are analyzed by means of electrospray ionization mass spectrometry (ESI). It is particularly preferred that the size of the fragments generated in step e) is adapted to the performance of the mass spectrometer.
  • EI electrospray ionization mass spectrometry
  • PCRs of a gene segment are carried out and the primers are gradually reset in such a way that the fragment size to be expected falls in at least one of these PCRs in the mass range detectable by mass spectrometry.
  • one of the PCR primers is gradually repositioned relative to the other around the maximum detectable mass range of the mass spectrometer.
  • step b) a PCR primer is provided with a chemical function so that the PCR product can be immobilized on a surface.
  • the PCR product produced in step b) is transferred to different reaction vessels and the surfaces of the reaction vessels are chemically such that the PCR product can be bound to them.
  • PCR products produced in step c) are transferred from different individuals into different reaction vessels prepared as described above.
  • an enzyme is used for step e) which forms a complex with a non-complementary base pair. It is particularly preferred that this enzyme is MutS.
  • the enzyme carries a label by which a complex can be illustrated.
  • the label is a fluorescent label, a chemiluminescent label, a mass day or a photochemically removable mass day.
  • an amplified DNA sample according to step c) in claim 1 which shows a difference to an amplified DNA sample in step b) in claim 1, is followed by a DNA sample itself in a second run of the method Step b) in claim 1 and is compared with all other DNA samples to be examined.
  • a preselection of the gene sections to be examined in detail by mass spectrometry is carried out by means of fluorescent labeling or chemiluminescent labeling of the immobilized DNA strand, the absence of which after carrying out steps d) and e) of claim 1 and a washing step the presence of indicates methylated cytosms in the examined genomic DNA section.
  • the present invention furthermore relates to a kit for carrying out the process according to the invention.
  • rens comprising DNA from at least two different individuals, tissues, cell lines or cells as well as reagents to show the variable methylation positions.
  • the invention further comprises completely methylated and / or demethylated DNA and reagents which are required for the detection of methylated cytosines in any DNA sample.
  • the method according to the invention serves to identify 5-methylcytosine positions in genomic DNA, which can be of very different origins.
  • genomic DNA is first treated chemically so that there is a difference in the reaction of the cytosine bases to the 5-
  • Methylcytosine bases results.
  • Possible reagents are e.g. B. disulfite (also called bisulfite or pyrosulfite), hydrazine and permanganate.
  • the genomic DNA is treated with disulfite in the presence of hydroquinone or hydroquinone derivatives, the cytosine bases being selectively converted to uracil after subsequent alkaline hydrolysis. 5-Methylcytosine remains unchanged under these conditions.
  • a certain section of the pretreated genomic DNA is now amplified in a polymerase reaction.
  • the polymerase chain reaction is used here.
  • the same section of another genomic DNA sample is then amplified equally.
  • the two amplificates are combined, which partially forms heteroduplexes.
  • this is done in such a way that one of the PCR primers is used for immobilization has a suitable function and that only one strand of the amplificate of the first sample is immobilized and then hybridization with the amplificate of the second sample takes place.
  • a multiplicity of different amplified products of the same nucleic acid section are hybridized in this way against the immobilized single strand from the amplified product of the first sample, which was distributed over many wells of a microtiter plate. A hybridization experiment can now be carried out in each well.
  • a procedure is carried out which leaves a detectable mark at the positions in which a base mismatch occurs in the heteroduplex.
  • this is carried out by chemical mismatch cleavage, which leads to a backbone fracture at the positions where a base mismatch occurs.
  • the fragments obtained in this way can be analyzed by any method that can show the size of DNA fragments. Such a method should ideally allow conclusions to be drawn about any position in the amplified nucleic acid section of the sample at which a base mismatch occurred in the heteroduplex.
  • Base mismatches in the heteroduplex are particularly present when cytosine was present in the DNA from a sample at this position, which was converted to uracil, but in the other 5-methylcytosine, which remained unchanged in the chemical pretreatment.
  • the method can be used for the comparison of two or more genomic DNA samples, in this case the analysis of the fragments only provides the differences in the methylation pattern between the two samples in each case amplified nucleic acid section. However, it is also possible to use a completely methylated or demethylated DNA as a reference. In this case, analysis of the fragments provides all 5-methylcytosine positions in the respective amplified nucleic acid section.
  • mass spectrometry is used for the fragment analysis.
  • the fragments can be analyzed in the MALDI mass spectrometer.
  • the solutions can be analyzed using electrospray ionization mass spectrometry (ESI).
  • ESI electrospray ionization mass spectrometry
  • the base mismatches - as an alternative to the analysis of fragments after a backbone cleavage in the heteroduplex - can also be detected by an enzyme that forms a complex with a non-complementary base pair.
  • this enzyme is MutS, which is a label, e.g. B. carries a fluorescent, chemiluminescent or mass marking.
  • the presence of base mismatch is detected by means of a fluorescence or chemiluminescence label.
  • an immobilized DNA strand from the amplificate of sample 1 is provided with a fluorescent label at the end not used for immobilization.
  • Heteroduplexes are formed with the amplificate of sample 2, using a chemical mismatch
  • the fluorescent label disappears after a denaturing washing step, if the strand is not cleaved, the label is retained. Only the amplificates that have been cleaved are subsequently examined in more detail by mass spectrometry.
  • the genomic DNA to be examined comes from a
  • the cell line or, if possible, only one cell is divided into two reaction vessels and the one half enzymatically either completely methylated on the cytosine or demethylated.
  • the enzyme is thermally inactivated and then both parts are put together again and treated with disulfite and then alkali. After purification, it is amplified by means of PCR.
  • Variant 1 The above method is carried out in such a way that a primer is used in the PCR which is functionalized in such a way that a simple and specific immobilization is made possible after the PCR. Immobilization takes place on beads or on the surface of a microtiter plate. This allows the simple separation of components of the polymerase and mismatch cleavage reactions. After the chemical mismatch cleavage reaction, the duplex is thermally denatured and the solution is pipetted off. The DNA fragments are applied from this solution to a reversed phase material and purified.
  • the fragments reveal a "ladder" of peaks, which indicates the methylated positions. Due to the symmetrical methylation at CpG positions, there are theoretically always two peaks per CpG, which originate from the sense and the antisense strand.
  • Variant 2 The reactions are carried out in solution and purification after the individual reaction steps is carried out, if necessary, using a reversed phase material.
  • Variant 3 Several individuals or cell types are examined in parallel. A reference DNA is completely demethylated and then treated with disulfite. she is amplified by PCR after purification. Again, a primer is used which has a function suitable for immobilization. The solution is distributed onto the wells of a microtiter plate and immobilized. This is followed by hybridization against the PCR products from samples also treated with disulfite, one for each well (see also detailed example with 97 individuals).
  • Variant 4 In the event that the mass spectrometer cannot cover the measuring range that would be required for the analysis of the entire PCR product for methylations, the area of interest can also be scanned step by step by performing several PCRs and one of the primers in each case the respective measuring range of the mass spectrometer is approached closer to the other. This means, for example, that only the area that lies between the primer of the respective and the next PCR to be shifted is always detected. The process can be combined with the other variants.
  • DNA from different individuals or cell lines is pooled and treatment with disulfite is carried out as described above. After alkaline hydrolysis of the bisulfite adducts and purification of the product DNA, this is amplified by means of PCR. It is then purified again and, after a few minutes of reannealing at 25 ° C., the PCR product is cleaved with Os0 4 at the positions using a C mismatch (chemical mismatch cleavage). A mismatch C against A always occurs if before the bisulfite Treatment only in some individuals where a methylated cytosine was present.
  • C mismatch chemical mismatch cleavage
  • any SNPs single nucleotide polymorphisms will also result in the cleavage of the DNA in this process. These must be differentiated from the methylation positions to be found, which is ensured by the method described above for finding all methylated cytosines.
  • the product DNA is now examined by mass spectrometry as described above. If the initially generated PCR product has a greater length than can be detected using the available technology on the part of mass spectrometry, it is possible that the fragments produced by the chemical mismatch cleavage cannot be detected. To avoid this, several PCRs can be carried out iteratively, i.e. one primer is always kept constant, while the other primer is always positioned in several steps closer to the other primer by the detection limit of the mass spectrometer (primer walking).
  • the product is purified and amplified using PCR.
  • One of the PCR primers is provided at the 5 'end with a chemical modification that is used for immobilization.
  • the product of this PCR is placed in the 96 chambers of a microtiter plate and the binding of the PCR products with the surface induced. Since only one primer is provided with the chemical modification for the binding, only one DNA strand binds to the surface. The plate is washed to remove binding chemistry reagents and complementary strands.
  • the plate with the reference DNA piece is now prepared.
  • the same genomic section is treated analogously with disulfite in each of the 96 other individuals and then amplified.
  • Two normal, unmodified primers of the same sequence as for the reference individual are used for this PCR.
  • the 96 PCR products are placed in the 96 wells of the prepared plate.
  • the complementary strands of the 96 individuals are hybridized to the reference DNA by heating and slow cooling (formation of the heteroduplex). Eliminate the 96 individuals and reagents from previous reactions.
  • An Os0 4 solution is added to each of the 96 wells, incubated and then a backbone break in a heteroduplex with a non-complementary base pair, one base of which is C, is induced with piperidine.
  • the heteroduplex that is to say in one of the individuals, has only one strand of methyl cytosine instead of one cytosine.
  • the cytosine of one individual was converted into an uracil before the PCR, which results in a mismatch in the heteroduplex with the counter strand of another individual.
  • the assay therefore does not directly yield all methylated cytosines of a genomic section, but only those that are variable between different individuals, tissues, cell lines or individual cells.
  • a good variant is to take the solution after melting the heteroduplex in a pipette tip that is equipped with a reversed-phase material.
  • the DNA products bind to this by forming hydrophobic interactions via their trialkylammonium counterions and can thus be cleaned of chemical mismatch cleavage reagents in several washing steps. With 30% acetonitrile, the DNA products can be detached from the reverse phase material. It makes sense to place the products directly on a prepared matrix on a MALDI target. After drying, the target is introduced into the mass spectrometer and the products are analyzed.
  • Example 5 Preselection by means of fluorescent labeling of gene segments relevant for methylation detection
  • the genomic DNA to be investigated is immobilized on beads or a correspondingly coated microtiter plate after the bisulfite reaction as described above with subsequent PCR amplification, in which one of the primers in turn has a function which is useful for the subsequent immobilization.
  • Fully demethylated DNA, treated as the sample DNA is used as the reference DNA and forms a heteroduplex with the immobilized sample DNA.
  • a single fluorescence-labeled base is appended enzymatically, for example with terminal transferase, to the 3 'ends of the product.
  • This method can also be used for simple, fluorescence-based detection of 5-methylcytosines in small gene segments, e.g. B. promoters can be used. However, only the statement can be made as to whether or not there are methylations in the region in question, but not how many and at which positions. However, there is a relatively small amount of experimentation and good parallelism.
  • the exact position of the methylcytosines can then be determined by mass spectrometry analogously to one of the examples given above.
  • the heteroduplexes immobilized in a microtiter plate are first brought together with a solution of MutS, to which a fluorescent dye is bound. Only the vessels in which MutS mismatch positions, which shows that after several washing steps the fluorescence is still detectable, are subsequently subjected to chemical mismatch cleavage and analyzed in the mass spectrometer. This saves time in the mass spectrometer and the cost of purification by avoiding the analysis of samples without demonstrable epigenetic information.

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Abstract

L'invention concerne un procédé d'identification de positions 5-Méthylcytosine dans l'ADN génomique. Ce procédé consiste a) à traiter chimiquement l'ADN génomique d'une cellule, d'une lignée cellulaire, d'un tissu ou d'un individu de telle façon que la cytosine et la 5-méthylcytosine réagissent différemment et que l'on obtienne dans les deux produits un comportement d'appariement des bases différent dans la double hélice; b) à amplifier la section d'acide nucléique de ce dernier par une réaction de polymérisation; c) à traiter la même section d'acide nucléique au moins d'une autre cellule, lignée cellulaire, d'un autre tissu ou individu ou d'un ADN de référence quelconque selon a) et b); d) à former des doubles hélices à partir d'au moins deux produits d'amplification obtenus en b) et c); e) à introduire un marquage repérable dans l'hétéroduplex par une réaction spécifique à des paires de bases non complémentaires.
EP99962069A 1998-11-19 1999-11-19 Procede d'identification de modeles de methylation de cytosine dans l'adn genomique Withdrawn EP1135526A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19853398 1998-11-19
DE19853398A DE19853398C1 (de) 1998-11-19 1998-11-19 Verfahren zur Identifikation von Cytosin-Methylierungsmustern in genomischer DNA
PCT/DE1999/003747 WO2000031294A2 (fr) 1998-11-19 1999-11-19 Procede d'identification de modeles de methylation de cytosine dans l'adn genomique

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JP (1) JP2002530118A (fr)
AU (1) AU757473B2 (fr)
CA (1) CA2351143A1 (fr)
DE (1) DE19853398C1 (fr)
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IS5916A (is) 2001-04-10
DE19853398C1 (de) 2000-03-16
WO2000031294A2 (fr) 2000-06-02
AU757473B2 (en) 2003-02-20
IL143176A (en) 2005-05-17
JP2002530118A (ja) 2002-09-17
IL143176A0 (en) 2002-04-21
AU1856500A (en) 2000-06-13
WO2000031294A3 (fr) 2000-10-19
CA2351143A1 (fr) 2000-06-02
NZ511638A (en) 2003-11-28

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