DE10204566A1 - Method for determining the methylation pattern of DNA - Google Patents

Abstract

A method is described for determining the methylation pattern in DNA sequences, in which unmethylated cytosine of the DNA sequence is hybridized with a detector DNA sequence, the hybrids being fixed in a spatially resolved manner on a surface. Guanine / uracil mismatches are then detected using substances which recognize such mismatches.

Description

The present invention relates to a method for determining the Methylation pattern of DNA especially using electronically addressable biochips.

The methylation of DNA plays an important role in nature. In particular the activity of genes is determined with the help of the methylation of individual cytosines that regulates DNA. To improve the activity and regulation of individual genes To be able to judge, there is a great interest in procedures with which simply have such methylations detected. For this reason meanwhile several methods have been developed to determine the Methylation rate and the methylation pattern in the DNA are suitable.

One method uses restriction enzymes that do not act as methylated sites Can recognize substrate (e.g. described in Analysis of DNA methylation and DNAse I sensitivity in gene-specific regions of chromatin; Ginder, Gordon; Methods Hematol. Vol. 20 1989 pp. 111-123). With these procedures you can however, only methylations that are linked to the Restriction interfaces occur.

In addition, there are other methods in which the Methylation of DNA sequences is determined (MethylLight: a high-througput assay to measure DNA methylation; Cindy A. Eads, Kathleen D. Danenberg, Kazuyuki Kawakami, et. al .; Nucleic Acids Rearch 2000 Vol. 28. No 8.). at However, this method must have one for each methylation position corresponding dye-labeled sample can be synthesized, which is technically very is complex.

In addition to the described biotechnological approaches, the chemical reactions with chloroacetaldehyde the methylation of DNA be determined quantitatively. The methylation is verified with this Method using a spectrometer in the range between 300 and 400 nm. (Quantification of 5-methylcytosine in DNA by the Chloroacetaldehyde Reaction; Biotechniques 27, 744-752 October 1999, Oakeley E. J, Schmitt F, Jost J. P.)

In an alternative method, the methylation pattern is made using bisulfite SSCP determined, where unmethylated cytosine is converted to uracil. With With the help of a DNA sequencer, the sequence of the DNA Sample to be determined (quantitative DNA methylation analysis by fluorescent ploymerase chain reaction single strand conformation polymorphism using an automated DNA sequencer; Hiromu Suzuki, Fumio Itoh, Minoru Toyota Tekefumi KiKuchi Electrophoresis 2000 21, 904-908).

The invention is therefore based on the object of a simple method for To provide determination of the methylation pattern of DNA sequences.

• a) Chemische Umwandlung von in Ausgangs-DNA enthaltenem nicht methylierten Cytosin zu Uracil, wobei die Sequenz der Ausgangs-DNA bekannt ist,
• b) Hybridisierung von einzelsträngigen, nach Schritt a) behandelter DNA mit einzelsträngigen Detektor-DNA-Sequenzen, die komplementär zur Sequenz der Ausgang-DNA oder deren Teilsequenzen sind, wobei die Guaninbasen gegen Adeninbasen ausgetauscht sein können,
• c) Fixierung von einzelsträngiger Detektor-DNA oder von einzelstrangigen nach Schritt a) behandelten DNA-Sequenzen vor oder während der Hybridisierung oder von Heteroduplices aus Detektor-DNA und nach Schritt a) behandelter DNA nach oder während der Hybridisierung, wobei die Fixierung ortsaufgelöst auf einem Träger erfolgt, so dass jeder Detektor-DNA-Sequenz ein bestimmter Ort zuzuordnen ist,
• d) Inkubation mit einem Guanin/Uracil- oder Adenin/Methylcytosin- Fehlpaarungen erkennenden Substrat und Detektion der Substratbindungen.

The task is solved by a process that comprises the following process steps:

• a) chemical conversion of unmethylated cytosine contained in the starting DNA to uracil, the sequence of the starting DNA being known,
• b) hybridization of single-stranded DNA treated according to step a) with single-stranded detector DNA sequences which are complementary to the sequence of the starting DNA or its partial sequences, it being possible for the guanine bases to be exchanged for adenine bases,
• c) fixation of single-stranded detector DNA or of single-stranded DNA sequences treated according to step a) before or during hybridization or of heteroduplexes from detector DNA and DNA treated after step a) after or during hybridization, the fixation being spatially resolved on a Carrier is carried out so that each detector DNA sequence can be assigned a specific location,
• d) Incubation with a guanine / uracil or adenine / methylcytosine mismatch substrate and detection of the substrate bonds.

For the purposes of the present invention, the term "starting DNA", understood a DNA or a DNA fragment with a known sequence, the Methylation pattern should be determined.

The term "detector DNA" includes DNA polynucleotides that Partial sequences of the starting DNA sequence are complementary, with preference all guanine bases are exchanged for adenine bases.

The concept of "spatially resolved" fixation means that a particular Detector DNA sequence can be assigned to a defined location on a support can. The loading of the carrier can be passive, for. B. using Pipetting devices or active, done via electronic addressing. method for the production of such carriers are known to the person skilled in the art. For the production Corresponding methods are electronically addressable carrier surface z. B. in Radtkey et al., Nucl. Acids Res. 28, 2000, e17, R.G. Sonowsky et al., Proc. Natl. Acad. Sci. USA, 1119-1123; 1994 P.N. Gilles et al., Nature Biotechnol. 17 365-370, 1999 described, the production itself by means of electronic addressing can be performed.

The starting DNA to be examined can e.g. B. an isolated from the cell nucleus, Be a gene or gene fragment whose nucleotide sequence is known. The Starting DNA can be further fragmented for better handling. The Fragmentation can be unspecific e.g. B. by shear forces or specifically z. B. by digesting a restriction. The DNA fragments thus obtained can then be denatured for chemical conversion.

The chemical conversion of unmethylated cytosine into the starting DNA sequences are preferably carried out using bisulfites, whereby Methylcytosine is not implemented in the reaction. The DNA treated in this way Sequence can now be hybridized with a detector DNA sequence, whereby the detector DNA sequence complementary to that chemically untreated Initial sequence can be. When hybridizing the chemically treated DNA sequence with the detector DNA sequence thus result Guanine / uracil mismatches at the sequence positions at which in the Starting sequence an unmethylated cytosine was present.

In a preferred embodiment, however, detector DNA Sequences uses their sequence complementarily to that chemically untreated starting sequence, the guanine bases being replaced by adenine bases are replaced. When hybridizing the detector DNA sequence with the chemically treated DNA sequence are thus adenine / methylcytosine mismatches receive. This method variant is particularly suitable for determining the Methylation pattern of low methylated DNA sequences, as they are mostly occur in mammals or plants. Sucker DNA has a methylation rate from 3 to 10% on that of plants up to 50%. In contrast, viruses point a degree of methylation of up to 100%, here also the direct Detection of the non-methylated cytosines via the corresponding Guanine / uracil mismatches can be beneficial.

It is also possible to carry out both process variants in parallel.

As detector DNA sequences z. B. synthetically accessible Polynucleotides, preferably with a length between 5 and 100, particularly preferably between 12 and 35 nucleotides can be used. The detector For this purpose, DNA sequences can be derived from the known starting DNA sequence derive directly. The individual detector DNA sequences can be different overlap, so that ultimately a mismatch occurs can be assigned to a specific location within the starting DNA sequence.

The mismatches can ultimately be identified with mismatching substances, such as B. mutS can be demonstrated.

The chemical conversion of unmethylated cytosine bases into DNA sequences is preferably carried out on single strands z. B. by denaturing double-stranded DNA samples can be obtained in alkaline. The converted DNA can then, if necessary, be further processed, that is separated, denatured again, neutralized, precipitated and desalted.

Methods for the chemical conversion of cytosine to uracil are e.g. B. described in "Bisulfite methylation analsis of single DNA strand", Tech. Tips Online 1998 BioMednet, Kimberely D Tremblay, Deaprtment of Biology, University of Pennsylvania, Philadelphia, PA 19104 USA and in "Promoter methylation analysis on microdissected paraffin-embedeed tissues using bisulfite treatment and PCR-SSCP ", Biotechniques 30: 66-72 (January 2001) pp. 66-72 the DNA to be converted containing methylated cytosine bases dissolved in water and then by adding a sodium bisulfite hydroquinone solution in alkaline implemented at elevated temperature with exclusion of light. To When the reaction is complete, the DNA obtained can be purified by chromatography become.

Hybridization of the chemically converted DNA nucleotides with the Detector DNA can take place on any surface to which the hybrid is attached can be bound locally. There are known ways such as B. the connection of nucleic acids via disulfide bridges Gold surfaces or the binding of biotinylated nucleic acids to avidin occupied surfaces available. The hybridization itself can be passive or actively electronically accelerated, as z. B. with a chip from the company Nanogen Inc. (San Diego / USA) is possible. The electronic addressing takes place by applying an electrical field, preferably between 1.5 V and 2.5 V with an addressing time between 1 and 3 minutes. Due to the electrical charge of the nucleotide sequences to be addressed becomes their Hike greatly accelerated by applying an electric field. The Addressing can take place in a spatially resolved manner, with different addresses Zones of the chip surface are addressed one after the other. You can send to the different locations different addressing and hybridization conditions can be set.

After hybridization, both the guanine / uracil and the Adenine / methylcytosine mismatches with mismatch-detecting substrates be detected. Suitable substrates are e.g. B. base mismatches binding proteins such as mutS or mutY, preferably from E.coli, T.thermophilus or T.aquaticus, MSH 1 to 6, preferably from S. cerevisiae, 51-Nuclease, T4- Containing endonuclease, thymine cosylase, cleavase or fusion proteins a domain of these base mismatch binding proteins.

The protein that recognizes mismatches can, in addition to dye-bearing, luminescent and fluorescent groups also polymeric markers (J. Biotechnol. 35, 165-189, 1994), metal markers, enzymatic or radioactive Markings or quantum dots (Science Vol 281, 2016, Sep 25, 1998) contain. The enzyme label can z. B. a direct enzyme coupling or be an enzyme substrate transfer or an enzyme complementation. to enzymatic labeling are especially suitable for chloramphenicol Acetyltransferase, the alkaline phosphatase, the luciferase or the Peroxidase.

In particular, substrate marking with dyes that absorb or emit light in the range between 400 and 800 nm is preferred. Among the fluorescent dyes suitable for labeling are especially Cy ^TM 3, Cy ^TM 5 (from Amersham Pharmacia), Oregon Green 488, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Bodipy 558/568 , Bodipy 650/665, Bodipy 564/570 (e.g. from Mobitec, Germany), S 0535, S 0536 (e.g. from FEW, Germany), Dy-630-NHS, Dy-635 -NHS, EVOblue30-NHS (e.g. from Dynomics, Germany), FAR-Blue, FAR- Fuchsia (e.g. from Medway, Switzerland), Atto 650 (from Atto Tech, Germany) , FITC or Texas Red. In addition to the directly labeled substrates, labeled antibodies can also be used which directly against mutS or against a fused peptide domain, such as. B. MBP are directed.

A defined nucleotide sequence is assigned to each location on the support surface can be obtained from the fluorescence signals obtained, in the cases in exactly one possible mismatch occurs at any location on the carrier can be translated directly into the corresponding methylation pattern. In the As a rule, however, there are several potential at each location on the carrier surface Mismatch positions (cytosines in the corresponding partial sequence of the Starting DNA) included. To determine the methylation pattern using the It is then expedient for fluorescence signals to overlap in their sequence To use detector DNA sequences. The evaluation then takes place after the Clustering analysis method (described in Eisen, MB, Spellman, PT, Brown P. O., Botstein D 1998, Cluster analysis and display of genome wide expression pattern, proc. Natl. Acad Sci USA 95, 14863-14868; Review: Discovering Pattern in Microarray Dat Molecular Diagnosis Vol. 5 No. 4 2000 Harry Burke. S349) or the "Principal Components analysis" method (described in Hilsenbeck SG, Friederichs WE, Schiff R., Statistical analysis of array expression data as applied to the problem of tamoxifen resistance J. Natl Cancer Institut 1999 91 453-459).

The specificity of measuring mismatches with mutS, a preferred one Mismatching substrate can be further enhanced by the addition of Surfactants or by adding BSA that indicate non-specific binding sites the hybrids are increased blocked. MutS also has the advantage that a measurement even under low salt conditions up to a Salt concentration of 10 mM can be done.

In order to further improve the reliability of the method by a Intermediate step any, fixed on the carrier after the hybridization step, single-stranded nucleotide sequences by adding a nuclease, preferred a mung bean nuclease or S1 nuclease.

With the method according to the invention, the methylation pattern of DNA Fragments with known sequences can be determined easily and quickly. In particular through the use of active, electronically addressable Chips can further reduce the measurement time and the reliability of the measurement, due to the very good electrical controllability of the hybridization become. The carriers once covered with detector DNA are also beyond reusable, the measured DNA can be easily dehybridized and removed from the Surface to be washed off. This enables a high sample throughput with an occupied carrier.

To explain the claimed method, the individual method steps are shown schematically in FIG. 1 as an example. Step I) shows the chemical conversion of starting DNA oligonucleotides. The oligonucleotides are shown in dashed lines, the left oligonucleotide containing a methylcytosine ( ^5m C), the right oligo in the same place an unmethylated cytosine (C). During the reaction, the unmethylated cytosine bases are converted into uracil. Subsequently, the oligonucleotides are applied to a chip surface, the detector having a DNA adenine base to the complementary to the positions ^5m C and C point on the detector-DNA oligonucleotide (step II)). Step III) shows the hybridization of the applied oligonucleotides with the detector DNA sequence, wherein in the presence of a methylcytosine at this position an mismatch occurs which can be detected with fluorescent-labeled mutS.

embodiments Detection of methylated C building blocks in DNA on an active electrical Chips and the enzyme MutS

In a first attempt it is shown that with the help of the claimed The detection of adenine / methylcytosine mismatches with fluorescence-labeled mutS succeeds as a substrate that recognizes mismatches. For hybridization.

For this, the oligonucleotides Seq. ID No. 1, 2 and 3 (0.05 µmol scale) diluted with water so that a final concentration of 10 picomoles / 1 µl is available. The aqueous solution was then dissolved in 50 mM histidine buffer 1: 100. Oligonucleotides used 1) Detector DNA with biotin at the 5 'end (sense + 2A (5'-biotinylated))

2) Model compounds to evaluate MutS binding (5'-CY3 labeled)

The methyl modified bases are marked as ^5m C.

Addressing on an electrically addressable chip with a hydrogel layer:
The biotinylated oligo was addressed at 2 V for 60 s on the electrodes of the chip (Nanogen Inc.) at two separate locations on the surface of the chip and fixed. The complementary DNA oligos were hybridized in a spatially resolved manner at 2 V for 120 s in histidine buffer. The chip was washed for 10 min with buffer A (20 mM Tris / HCl pH, 7.6, 50 mM KCl, 5 mM MgCl2. 0.01% Tween / 20, 1% BSA).

0.5 µg Alexa Fluor 647 (from Mobitec, Germany), labeled mutS were taken up in 100 ul buffer A. The chip is released with the mutS for Incubated for 45 minutes. After 45 minutes, the chip with 10 ml of buffer A without BSA rinsed and then determined the fluorescence with a scanner.

The result is shown in Fig. 2. The fluorescence of two measurements taken independently of one another was at the place where the hybridization of Seq. ID No. 1 and 2 led to an adenine / methylcytosine mismatch (bar 1 in Fig. 2) at 60.3 (spot 1) and 55.7 (spot 2) the fluorescence was at the locations where there was no mismatch at 47.9 (spot 3) and 29.5 (spot 4). In FIG. 2, the corresponding mean values of two measurements are shown. Chemical modification of cytosine building blocks to uracil building blocks 10 µg or 5 µg or 1 µg of an oligonucleotide Seq. ID No. 4

(Starting DNA oligo that was chemically treated (5'-CY3 labeled))
with a methylcytosine building block were dissolved in 100 ul water. Then 10 μl of 3 N aqueous NaOH solution were added. The 110 ul solution was 30 min. heated to 42 ° C and then taken up in 1020 µl 40.5% sodium bisulfite (pH 5) and 60 µl 10 mM hydroquinone solution. After adding a further 10 μl of water, the mixture was heated at 55 ° C. for 16-18 hours with the exclusion of light. Then 500 µl of the corresponding solution were pipetted onto a NAPS column (Pharmacia), which was conditioned according to the manufacturer's instructions. The corresponding oligonucleotide was eluted with 800 ul water. The reaction is completed by adding 3 M NaOH to the oligonucleotide fraction obtained to a final NaOH concentration of 0.3 M at 37 ° C. The chemically converted DNA oligonucleotides obtained are then purified again by chromatography on a NAPS column and desalted. 50 ul of the desalted solution were diluted with 50 ul 100 mM histidine solution. DNA oligonucleotides are obtained whose unmethylated cytosine bases have been converted into uracil.

Preparation of the 40.5% sodium bisulfite solution (Sigma. S-8890. 8.1 g / 20 ml): For this purpose, the solid substance was dissolved in 17 ml of cold water and washed with 10 N NaOH Solution adjusted to a pH of 5 and then made up to 20 ml. A 10 mM (Sigma H-9003) solution was used as the hydroquinone solution. (The chemical conversion of cytosine to uracil is described in e.g. "Bisulfite methylation analsis of single DNA strand", Tech. Tips Online 1998 BioMedNet; Kimberely D Tremblay, Deaprtment of Biology, University of Pennsylvania, Philadelphia, PA 19104 USA and in "Promoter methylation analysis an microdissected paraffin-embedeed tissues using bisulfite treatment and PCR- SSCP ", Biotechniques 30: 66-72 (January 2001) pp. 66-72)

Non-specific labeling of mutS with Alexa Fluor 647

For conjugation, the corresponding Alexa Fluor 647 succinimidyl ester is non-specifically covalently linked to protein lysine residues via a nucleophilic substitution reaction in accordance with the FluoroLink ^TM product specification protocol, Amersham Pharmacia Biotech, Little Chalfont, UK. The fluorescence labeling of mutS takes place by incubation with a large excess of fluorophore under strongly basic conditions (0.1 M Na ₂ CO ₃ , pH 9.3). The fluorescence-labeled mutS obtained is purified by gel permeation chromatography. SEQUENCE LISTING

Claims (7)

1. A method for determining the methylation pattern of DNA comprising the following method steps: a) chemical conversion of the starting DNA with a known sequence, the unmethylated cytosine contained therein being converted into uracil, b) hybridization of single-stranded DNA treated after step a) with single-stranded detector DNA sequences which are complementary to the starting DNA sequence or partial sequences thereof, it being possible for the guanine bases to be replaced by adenine, c) fixation of single-stranded detector DNA sequences or of single-stranded DNA sequences treated according to step a) before or during hybridization or of heteroduplexes from detector and DNA sequences treated according to step a) after or during hybridization, the fixation is spatially resolved on a carrier, so that each detector DNA sequence can be assigned a specific location, d) Incubation with a guanine / uracil or adenine / methylcytosine mismatch substrate and detection of the substrate bonds. 2. The method according to claim 1, characterized in that the Hybridization takes place electronically accelerated. 3. The method according to any one of claims 1 or 2, characterized in that the fixation of single-stranded detector DNA sequences or single-stranded DNA sequences treated according to step a) before or during hybridization or from heteroduplexes from detector and after step a) treated DNA sequences after or during the Hybridization with local resolution on an electronically addressable Surface takes place. 4. The method according to any one of claims 1 to 3, characterized in that the chemical conversion of the unmethylated cytosine with a bisulfite. 5. The method according to any one of claims 1 to 4, characterized in that the single-stranded nucleotide sequences fixed on the support, the are not hybridized, can be degraded by adding a nuclease. 6. The method according to any one of claims 1 to 5, characterized in that as detector DNA, DNA sequences with a length of 5 to 100 Nucleotides can be used. 7. The method according to any one of claims 1 to 6, characterized in that overlapping DNA sequences are used as detector DNA.