DE19853398C1 - Identification of 5-methylcytosine positions in genomic DNA by chemical modification, amplification and heteroduplex formation - Google Patents

Abstract

A method for identifying 5-methylcytosine positions in the genomic DNA of a cell, cell line, tissue or individual is new and comprises: (1) chemically modifying the DNA by a treatment in which cytosine and 5-methylcytosine react differently, so that the cytosine and 5-methylcytosine positions will have different base-pairing properties; (2) amplifying a segment of the DNA by means of a polymerase reaction; (3) treating the same segment from at least one other cell, cell line, tissue or individual or from reference DNA as in (a) and (b); (4) forming heteroduplexes from the amplification products; and (5) detectably marking the heteroduplexes by a reaction specific for noncomplementary base pairs. Independent claims are also included for the following: (1) a kit for carrying out the method, comprising: (a) DNA from at least two maximally different individuals, tissues, cell lines or cells; and (b) reagents for detecting the variable methylation positions; (2) a kit for carrying out the method, comprising: (a) completely methylated and/or demethylated DNA; and (b) reagents for detecting methylated cytosines in any DNA sample.

Description

1. Brief description of the field of the invention

The genetic information obtained by complete sequencing of genomic DNA Base sequence is obtained, the genome of a cell describes only incompletely. 5- Methylcytosine nucleobases by reversible methylation of DNA in the cell arise, are an epigenetic information carrier and serve, for example, for Regulation of promoters. The methylation state of a genome represents that current status of gene expression, similar to an mRNA expression pattern. The subject of the present patent is a method for inexpensive and parallelizable detection of epigenetic information carriers in the form of 5- Methylcytosine bases in genomic DNA.

2. State of the art

5-methylcytosine is the most common covalently modified base in DNA more eukaryotic Cells. For example, it plays a role in the regulation of transcription, genomic Imprinting and tumorigenesis. Identification of 5-methylcytosine as an ingredient genetic information is therefore of considerable interest. 5-methylcytosine positions cannot be identified by sequencing, however, because 5-methylcytosine has the same base pairing behavior as cytosine. Unfortunately one goes PCR amplification the epigenetic information that the 5-methylcytosines carry completely lost, and there is no procedure for this information by one To obtain the amplification step.

Several methods are known to solve these problems. Usually a chemical Reaction or enzymatic treatment of the genomic DNA is carried out as a result of which the cytosine can be distinguished from the methyl cytosine nucleobases. A common one The method is the conversion of genomic DNA with disulfite (also as bisulfite or Pyrosulfite), which after alkaline hydrolysis in two steps to one Conversion of the cytosine bases into uracil leads (Shapiro, R., Cohen, B., Servis, R. Nature 227: 1047 (1970). 5-Methylcytosine remains unchanged under these conditions. The  5-Methylcytosine remains unchanged under these conditions. The conversion from C to U leads to a change in the base sequence, which now results from sequencing have the original 5-methylcytosine determined (only these still provide a band in the C- Track).

An overview of the other known options for detecting 5-methylcytosine, can be found together with the associated literature in the following review article: Rein, T., DePamphilis, M.L., Zorbas, H., Nucleic Acids Res. 26, 2255 (1998).

DD 293 139 A5 describes a method for characterization described certain DNA sequences, in which the DNA molecules whose unmethylated recognition sites by cut an appropriate restriction endonuclease are to be in the reaction mixture with a second, un methylated DNA species (especially oligonucleotide Duplexes containing the recognition site) incubated.

WO 97/46705 A1 discloses a method for detection a methylated, CpG-containing nucleic acid, wherein the sample containing nucleic acid with a reagent which unmethylated cytosine modifi adorned with the CpG-containing nucleic acid in the sample amplified using CpG-specific oligonucleotide primers, the oligonucleotide primer between modified me thylated and unmethylated nucleic acids under separates and detects the methylated nucleic acids.

US 5,824,471 A1 also describes a method for loading detection of deviations between two nucleic acids strands, with a variety of duplexes from the two Strands or their parts formed and this duplex with a first and a second different bacts riophage resolvase is contacted and wherein one then determines from which bacteriophage resolvase the duplex is split, thereby the sub differences are determined.

However, it is not always necessary to actually have the entire sequence of a gene or Genetic section to determine how this is the goal in sequencing. This is especially the case when there are few 5-methylcytosine positions within a longer one Base sequence to be scanned for a variety of different samples. The Sequencing provides redundant information on a large scale and is also very good expensive. This is the case even if the sequence is already known and only methylation positions should be shown. It is also conceivable that in In some cases, only the differences in the methylation pattern between different genomic DNA samples are of interest and that to identify one Numerous matching methylated positions as well as sequencing can be dispensed with. So far, there is no answer to the questions listed here Process that inexpensively produces the desired ones without sequencing each individual sample Delivers results.

Sequence information also has to be determined less and less because the Genome projects, the goal of which is the complete sequence of different organisms, quickly progress. Only about 5% of the human genome is currently fully sequenced, however, come now because other genome projects are coming to an end and thereby Free sequencing resources, an additional 5% every year. With the completion of the Sequencing of the human genome is expected by 2006.

Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) 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 converted into an im UV absorbing matrix embedded. The matrix is insulated by a short laser pulse Vacuum evaporates and the analyte is thus transported unfragmented into the gas phase. A The applied voltage accelerates the ions into a field-free flight tube. Because of your  converted. This technology is currently capable of a mass range from 1,000 to 4,000 Da to distinguish molecules with a mass difference of 1 Da. Through the However, most biomolecules are already about 5 Da in natural distribution of isotopes wide. Technically, this mass spectrometric method is excellent for analysis of biomolecules. Products that need to be analyzed reasonably need that to be distinguished, at least 5 Da are apart. In this mass range 600 molecules could be differentiated. In the range between 4,000 and 100,000 Although isotope resolution is no longer achieved, this range can also be used. Recently, the use of an infrared (IR) laser is coupled with the MALDI analysis from DNA has been described (Berkenkamp, S., Kirpkar, F. and Hillenkamp, F. 1998. Infrared MALDI mass spectrometry of large nucleic acids. Science. 281: 260-262). Through this Combination made it possible to make DNA fragments up to 2,500 bases in size detect.

Chemical mismatch cleavage is a method by which small differences between two DNA single strands can be shown (Cotton, R.G.H., Rodriguez, N.R. and Campbell, R. D. 1988. Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. UNITED STATES. 85: 4397-4401; Cotton, R.G.H. 1993. Current methods for mutation detection. Courage. Res. 285: 125-144; Saleeba, J.A. and Cotton, R.G.H. 1993. Chemical cleavage of mismatch to detect mutations. Methods in Enzymology. 217: 286-295; Smooker, P.M. and Cotton, R.G.H. 1993. The use of chemical reagents in the detection of DNA mutations. Mutations Res. 288: 65-77). The chemical reactivity of C and T towards Osmium tetroxide and of C versus hydroxylamine is increased when not with theirs respective complementary bases are paired. Through the subsequent treatment with Piperidine breaks the strand of nucleic acid at the modified position.

Another possibility of non-complementary base pairs in heteroduplex DNA To show is to use enzymes like MutS that are not complementary Bind base pairs (Smith, J. and Modrich, P. 1996. Mutation detection with MutH, MutL and MutS mismatch repair proteins. Proc. Natl. Acad. Sci. USA 93: 4374-4379; Parsons, B.L. and Heflich, R.H. 1997. Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA. Courage. Res. 374: 277-285).

3. Task

There is currently no fast, inexpensive and automatable method of finding of methylated cytosines in genomic DNA. Such a procedure is of great importance Interest, because different methylation patterns in various ways Characterization of cell types and thus for the diagnosis and classification of Diseases (such as tumors) can be used as well Example of studies of cell differentiation could be used.  

4. Description

The method is used to identify 5-methylcytosine positions in genomic DNA, can be of various origins. The genomic DNA first becomes chemical treated 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. In a preferred variant of the The genomic DNA is disulfite-treated in the presence of hydroquinone or Hydroquinone derivatives treated, selectively after subsequent alkaline hydrolysis the cytosine bases are converted to uracil. 5-methylcytosine remains among these Conditions unchanged. After a purification process, the separation of the excess disulfite is used, a certain section of the pretreated is now amplified genomic DNA in a polymerase reaction. In a preferred variant of the process, the polymerase chain reaction is used here. Then will the same section of another genomic DNA sample is equally amplified. The the two amplificates are combined, resulting in partial heteroduplexes form. In a preferred variant of the method, this is done in such a way that one of the PCR primer has a function suitable for immobilization and that only one strand the amplificate of the first sample is immobilized and then a hybridization with the amplificate of the second sample. In a further preferred variant, a Many different amplificates of the same nucleic acid section in this way and Way against the immobilized single strand from the amplificate of the first sample hybridized, which was distributed over many wells of a microtiter plate. In every well can a hybridization experiment can now be carried out.

After hybridization, a procedure is carried out at the positions in which a Base mismatch in which heteroduplex occurs, leaving a detectable mark. In A preferred variant of the method is chemical mismatch cleavage performed at the positions where base mismatch occurs Backbone fracture leads. The fragments thus obtained can be any Analyze methods that can reveal the size of DNA fragments. Such one Ideally, the method should draw conclusions about each position in the amplified Allow nucleic acid portion of the sample where there is a base mismatch in the Heteroduplex occurred. Base mismatches in the heteroduplex are especially then present if cytosine was present in the DNA from a sample at that position  was converted into uracil, but in the other 5-methylcytosine, which is used in chemical Pretreatment remained unchanged. The method can be used to compare two or two several genomic DNA samples are used, in this case the analysis of the Fragments only the differences in the methylation pattern between the two samples in the respective amplified nucleic acid section. But it is also possible to complete one to use enzymatically methylated or demethylated C as a reference. In this The analysis of the fragments provides all 5-methylcytosine positions in the respective case amplified nucleic acid section.

In a particularly preferred variant of the method, mass spectrometry for the Fragment analysis used. The fragments can be cleaned in the MALDI mass spectrometers can be analyzed. Alternatively you can use the solutions Electrospray ionization mass spectrometry (ESI) can be analyzed. Depending on The performance of the method and the instrument used may require to examine the nucleic acid section in question in several substeps by: several PCRs, a primer is gradually repositioned and thus different Partial certificates result ("primer walking").

In a variant of the method, the base mismatches - as an alternative to the analysis of Fragments after a backbone cleavage in the heteroduplex - also by an enzyme can be detected, which forms a complex with a non-complementary base pair. In a preferred variant, this enzyme is MutS, which is a label, e.g. Legs Fluorescence, chemiluminescent or mass marking.

In a further variant of the method, the presence of base mismatch, that is to say in this case also of relevant information in the amplified nucleic acid section, is detected by a fluorescent or chemiluminescent label. In a preferred variant of the method, 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 and are subjected to a chemical mismatch cleavage. If there is a backbone cleavage on the immobilized strand, 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.

5. Examples 5.1. Procedure for finding all methylated cytosine positions

The genomic DNA to be examined comes from a cell line or, if possible, only one The cell is divided into two reaction vessels and one half enzymatically either completely methylated or demethylated on cytosine. The enzyme is thermally inactivated and then both parts are put together again and with disulfite and subsequently treated with alkali. After purification, it is amplified by means of PCR.

The chemical mismatch cleavage now carried out, which is specific for C mismatches, leads to a cleavage at the positions of a respective heteroduplex at which one originally methylated C was present in the event of complete demethylation of the half of the genomic sample was performed. Conversely, there is a split all originally non-methylated positions if complete methylation of the half of the genomic sample was previously performed. For security, too Both methylation and demethylation are carried out in this reference In this case, however, these must be used separately in two PCRs.

Variant 1: The above procedure is carried out using a primer in the PCR is functionalized so that a simple and specific after the PCR Immobilization is made possible. 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 chemical mismatch cleavage 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 cleaned.

In the mass spectrometer, the fragments give a "ladder" of peaks that point to the methylated positions can be closed. It falls due to the symmetrical methylation at CpG positions theoretically always two peaks per CpG, each from the sense and from the antisense strand.

Variant 2: The reactions are carried out in solution and a purification after If necessary, individual reaction steps take place in each case via 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. It will follow  Purification amplified by PCR. Again, a primer is used, the one function suitable for immobilization. The solution is on the wells one Microplate distributed and immobilized. Then hybridization takes place 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 gradually scanned by multiple PCRs be carried out and one of the primers around the respective measuring range of the Mass spectrometer is closer to the others. For example only covers the area between the primer to be shifted and the next PCR. The process can be combined with the other variants.

5.2. Method for finding positions with variable cytosine methylation

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 OsO ₄ at the positions using a C mismatch (chemical mismatch cleavage). A mismatch C against A occurs whenever a methylated cytosine was only present in a few individuals before the bisulfite treatment. At the same time, any SNPs (single nucleotide polymorphisms) will lead to 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 that available existing technology can be demonstrated by mass spectrometry, it is possible that the fragments produced by the chemical mismatch cleavage are undetectable are. To avoid this, several PCRs can be performed iteratively, that is one primer is always kept constant, while the other primer is always in several  Steps each closer to the other by the detection limit of the mass spectrometer Primer is positioned (primer walking).

5.3. Procedure with 97 individuals

A genomic section of an individual (reference individual) is treated with disulfite and thus the cytosines are converted into uraciles after subsequent alkaline hydrolysis of the bisulfite adduct. The methyl cytosines remain untouched in this reaction sequence. 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 to the surface is 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 OsO ₄ solution is added to each of the 96 wells, incubated, and then a backbone break is induced with piperidine in a heteroduplex with a non-complementary base pair, one of which is base C. This is always the case if the heteroduplex, that is to say in one of the individuals, has only one strand of methyl cytosine instead of one cytosine. In this case, only 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. The heteroduplex is melted by heating and the solution is transferred to a mass spectrometer.

5.4. Transfer the solution to the mass spectrometer

A good variant is the solution after melting the heteroduplex into one Pick up pipette tip, which is equipped with a reversed phase material. The DNA Products bind to it by interfering with their hydrophobic interactions Trialkylammonium counterions form and can thus in several washing steps of Chemical mismatch cleavage reagents are cleaned. With 30% acetonitrile the DNA products are released from the reverse phase material. It is a good idea to transfer the products directly to a prepared matrix on a MALDI target. To After drying, the target is introduced into the mass spectrometer and the products analyzed.

5.5. Preselection using fluorescent labeling for methylation detection relevant gene segments

The genomic DNA to be examined is named after the bisulfite as described above Reaction with subsequent PCR amplification, in which one of the primers is used for subsequent immobilization function, on beads or a corresponding coated microtiter plate immobilized. Fully demethylated DNA like that Sample DNA treated, is used as reference DNA and forms with the immobilized sample DNA a heteroduplex. Then enzymatic, for example with terminal transferase, a single fluorescence-labeled base at the 3 'ends of the product attached. The "Chemical Mismatch Cleavage" reaction carried out below leads to in the event that there is a C / A mismatch in the product, to split the immobilized strand so that after thermal dehybridization all Fluorescence markings are removed in the subsequent washing step. So it was If methylation is present within the amplificate, no fluorescence occurs in the Microtiter plate or on the bead more. This is only retained if there is no mismatch is present and there are therefore no 5-methylcytosines in the gene segment in question. At the procedure must be considered that z. B. SNPs give a false positive signal can.

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. But it can only the statement can be made as to whether there are methylations in the region in question or  not, but not how many and at what positions. However, this is a relatively small one experimental effort and a good parallelizability.

In the gene segments classified as relevant, one of the above can then be analogously Examples given the exact position of the methylcytosine by mass spectrometry be determined.

5.6. Preselection of heteroduplexes with mismatches

The heteroduplexes immobilized in a microtiter plate are first of all with a MutS solution to which a fluorescent dye is bound. Only the vessels in which MutS has accumulated at base mismatch positions, which is shows that after several washing steps the fluorescence is still detectable, are subsequently subjected to chemical mismatch cleavage and in Mass spectrometer analyzed. This saves time in the mass spectrometer and the cost saved for purification by analyzing samples with no detectable epigenetic information is avoided.

Claims (29)

1. A method for identifying 5-methylcytosine positions in genomic DNA, characterized in that the following method steps are 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 the duplex results in a different base pairing behavior of the two products,
• b) the same nucleic acid section is amplified by means of a polymerase reaction,
• c) the same nucleic acid section of at least one further cell, cell line, tissue or individual or any reference DNA is treated in accordance with points a) and b),
• d) heteroduplexes are formed from the at least two amplificates of points b) and c),
• e) by means of a reaction which is specific for non-complementary base pairs, an identifiable label is introduced into the heteroduplex.

2. The method according to claim 1, characterized in that it for the identification of Differences in the cytosine methylation pattern between different cells, cell lines, Tissues and individuals is applied and only shows positions in which the cytosine Methylation between different cells, cell lines, tissues or individuals is variable. 3. The method according to claims 1 to 2, characterized in that in step a) of Claim 1. a disulfite (bisulfite, pyrosulfite) as a reagent for the selective conversion of Cytosine is used in uracil, whereby 5-methylcytosine remains unchanged. 4. The method according to any one of claims 1 to 3, characterized in that genomic DNA of several individuals, tissues, cell lines or cells in step b) of claim 1 are amplified together. 5. The method according to any one of claims 1 to 3, characterized in that genomic DNA of several individuals, tissues, cell lines or cells are amplified separately and then treated together according to point e) of claim 1.   6. The method according to any one of claims 1 to 5, characterized in that by education of heteroduplexes from the DNA of various individuals, tissues, cell lines or cells base mismatches occur at the positions where a 5- in the genomic DNA Methylcytosine was localized. 7. The method according to claim 1, characterized in that in point d) by forming Heteroduplexes with a fully methylated reference DNA at the positions Base mismatches occur where there was cytosine in the genomic DNA. 8. The method according to claim 1, characterized in that in point d) by forming Heteroduplexes with a completely demethylated reference DNA at the positions Base mismatches occur in which 5-methylcytosine is found in the genomic DNA found. 9. The method according to any one of claims 6 to 8, characterized in that the Base mismatches using "chemical mismatch cleavage" complementary positions) to a specific or sufficiently selective Spine splitting at these positions. 10. The method according to any one of claims 6 to 8, characterized in that the DNA the base mismatch is cleaved enzymatically specifically or sufficiently selectively. 11. The method according to any one of claims 1 to 10, characterized in that in point e) of claim 1. DNA fragments are obtained, the size of which allows conclusions to be drawn Cleavage positions and thus on the position of the methylcytosine and / or between different individuals, tissues, cell lines or cells Methylation positions allowed. 12. The method according to claim 11, characterized in that the analysis of the size (Molecular weights) of the DNA fragments is carried out by means of mass spectrometry. 13. The method according to claim 12, characterized in that the fragments by means of matrix assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI) analyzed become.   14. The method according to claim 12, characterized in that the fragments by means of Electrospray ionization mass spectrometry (ESI) can be analyzed. 15. The method according to claims 13 and 14, characterized in that the size of the in point e) of claim 1 generated fragments of the performance of Mass spectrometer is adjusted. 16. The method according to claim 15, characterized in that several PCRs one Genabes are executed and the primers are gradually reset so that the expected fragment size in at least one of these PCRs in the means Mass spectrometry demonstrable mass range falls. 17. The method according to claim 16, characterized in that one of the PCR primers step by step relative to the maximum detectable mass range of the mass spectrometer is repositioned to the other. 18. The method according to any one of claims 1 and 2, characterized in that in Process step b) a PCR primer is provided with a chemical function so that the PCR product can be immobilized on a surface. 19. The method according to any one of claims 1 and 2, characterized in that the in Process step b) produced PCR product is placed in different reaction vessels and the surfaces of the reaction vessels are of a chemical nature such that the PCR product can be bound to it. 20. The method according to any one of claims 1 and 2, characterized in that in Process step c) produced PCR products of different individuals in different prepared reaction vessels are given according to claim 19. 21. The method according to any one of claims 1 and 2, characterized in that for the Process step e) an enzyme is used which is complementary to a non-complementary one Base pair forms a complex.   22. The method according to claim 21, characterized in that this enzyme is MutS. 23. The method according to claim 21, characterized in that the enzyme is a label through which a complex can be illustrated. 24. The method according to claim 21, characterized in that the marking is a Fluorescent label, a chemiluminescent label, a mass tag or a is photochemically removable mass day. 25. The method according to claims 1-24, characterized in that an amplified DNA sample of group c) in claim 1, which differs from an amplified one DNA sample of group b) in claim 1 in a second round of the method even a DNA sample of group b) in claim 1 and with all others investigating DNA samples is compared. 26. The method according to claims 1-25, characterized in that a preselection the gene segments to be examined in detail by mass spectrometry via a Fluorescent labeling or chemiluminescent labeling of the immobilized DNA Strands occurs, the absence of which after performing steps d) and e) of claim 1 and a washing step the presence of methylated cytosines in the examined indicates genomic DNA section. 27. The method according to claims 1-25, characterized in that a preselection the gene segments to be examined in detail by mass spectrometry via a more unspecific variant according to claims 20 to 23. 28. Kit for the method according to claims 1-26, consisting of the DNA of at least two different individuals, tissues, cell lines or cells as well as Reagents to show the variable methylation positions. 29. Kit for the method according to claims 1-26, consisting of completely methylated and / or demethylated DNA and reagents used for the detection of methylated cytosines are required in any DNA sample.