WO2006050864A1 - Method for identifying methylated cytosine - Google Patents

Method for identifying methylated cytosine Download PDF

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WO2006050864A1
WO2006050864A1 PCT/EP2005/011831 EP2005011831W WO2006050864A1 WO 2006050864 A1 WO2006050864 A1 WO 2006050864A1 EP 2005011831 W EP2005011831 W EP 2005011831W WO 2006050864 A1 WO2006050864 A1 WO 2006050864A1
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enzyme
deaminase
dna
nucleic acid
analysis
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PCT/EP2005/011831
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German (de)
French (fr)
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Jens Burmeister
Walter Weichel
Reinhard Koch
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Bayer Technology Services Gmbh
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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 invention relates to a method for the detection of 5-methyl-cytosine in DNA sequences and test kits containing those reagents that are needed to carry out the described assays.
  • 5-methyl-cytosine has a wide field of application, e.g. in human or veterinary diagnostics, in the food sector, in environmental analysis, in crop protection, in biochemical or pharmacological research as well as in forensic medicine.
  • the detection of 5-methyl-cytosine is e.g. by determining the length of specific restriction fragments in a fragment mixture (E.M. Southern, J. Mol. Biol. 1975, 98, 503-517).
  • the restriction endonucleases used here can distinguish the bases cytosine and 5-methyl-cytosine within their recognition sequence. This method is expensive (analysis by electrophoresis or similar) and not suitable for analyzing long sequence sections with frequent methylations.
  • only methylated cytosines that are within the recognition region of a restriction endonuclease can be analyzed using this method.
  • Selective base conversion by treatment with sodium bisulfite (Shapiro, R., et al., J. Am. Chem. Soc.
  • cytidine deaminases The enzyme family of cytidine deaminases has long been known. However, it was not until 2002/2003 that cytidine deaminases were described, which not only deaminate the nucleobase, the nucleoside or the nucleotide, but also catalyze the deamination of sequence-internal cytosines into DNA single strands (Bransteitter, R., et al., Proc. Natl. Acad., See, USA, 2003, 100: 7, 4102-4107, P. Pham et al. , Nature, 2003, 424, 103-107).
  • AID activating-induced cytidine deaminase
  • cytosine and 5-methyl-cytosine have also been described (R. Bransteitter et al., Proc. Natl. Acad. Sci., USA, 2003, 100: 7, 4102-4107 ).
  • WO 98/45472 a method for the screening of inhibitors of cytidine deaminase is described.
  • the application describes the use of short, synthetic RNA sequences which are incubated with cytidine deaminase in the presence of the potential inhibitors. Primer-mediated single base extension was used to assess whether conversion C-> U had occurred or whether deaminase activity was inhibited.
  • the present invention is therefore based on the object to provide a method for gentle, specific conversion of non-methylated cytosines in uracil. Ideally, it may be used in combination with detection methods known to those skilled in the art such as e.g. PCR, sequencing, hybridization in solution or on arrays for analysis of methylation patterns.
  • the solution according to the invention of the described object consists in the substitution of the Bisulf ⁇ t treatment by a specific, enzymatically catalyzed deamination of the unmethylated
  • Cytosine for the enzymatic treatment according to the invention a deaminase, preferably a
  • Cytidine deaminase particularly preferably a cytidine deaminase selected from the group of
  • APOBEC enzymes most preferably AID or APOBECl.
  • a deaminase which has been prepared by biotechnological methods, as known to the person skilled in the art, by altering a naturally occurring deaminase, can also be used for catalyzing the deamination.
  • the isolation of the naturally occurring deaminase can also be used for catalyzing the deamination.
  • Deaminase is carried out by methods known to those skilled in the art and is described, for example, in R.
  • methods for the detection of methylated cytosines containing the steps A) Providing an enzyme that selectively catalyzes the deamination of non-methylated cytidine to uracil.
  • a mixture of different deaminases can also be used for the deamination step A).
  • differences in the substrate specificity of various deaminases can be compensated.
  • the target DNA is incubated with the enzyme (s) under conditions in which the DNA to be deaminated is single-stranded.
  • This can be done, for example, by thermal denaturation of the DNA (eg at 90-95 0 C) and then rapid cooling down to substantially maintain the single-stranded form (eg at 4 0 C) (see, for example: MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag , New York, 1998, p. 9 ff.).
  • the enzymatic deamination can also be carried out under conditions under which the DNA, but not the enzyme, is denatured.
  • Low ion concentration, in particular low cation concentration and dilution of the DNA target solution can also slow the reassociation of denatured DNA.
  • formamide is added to the DNA target solution, the melting point of the DNA is also lowered, slowing down reassociation.
  • proteins that bind to single-stranded DNA the DNA can be obtained in its single-stranded form.
  • An example of such a DNA-binding protein is the single-stranded DNA binding protein sold, for example, by Promega, Madison, WI, USA.
  • the above-mentioned methods can be used individually or in suitable combinations with one another in order to make the DNA substrate available for the deaminase reaction.
  • this step is carried out by incubating the deaminase or the mixture of deaminases with the DNA target under respectively optimized temperature and buffer conditions which ensure the single-strandedness of the target while optimally converting the enzyme / enzyme mixture.
  • the DNA sequence is analyzed, wherein the conversion of a cytosine position into uracil indicates that there was no methylation at this sequence position, while unchanged cytosine positions carried methylation. In this way, the methylation pattern of the DNA can be analyzed.
  • the analysis of the DNA sequence is carried out, for example, by hybridization-based or enzymatic detection methods known to the person skilled in the art.
  • the analysis of the sequence modified by the deaminase treatment by enzymatic methods is preferably carried out by a PCR reaction, for example as a methylation-specific PCR (for a review see, for example: C. Dahl, Biogerontology 2003, 4, 4, 233 -250) or methylation-specific real-time PCR (example: YM Lo et al., Cancer Research 1999, 59, 3899-3903).
  • the modified nucleic acid is amplified with two sets of primers and associated real-time probes, the one set, for example, only binds if all cytosines are unmethylated and the second set binds, for example, only if the sequence is methylated at known positions.
  • the two different real-time probes can be distinguished by two different fluorophores (e.g., fluorescein and rhodamine). During real-time PCR, the increase in fluorescence due to the target amplification is determined.
  • the number of PCR cycles that is needed to exceed a specified threshold of fluorescence is defined as the so-called Ct value (Cycle threshold).
  • Ct value can be set via a calibration curve in relation to the target concentration used and thus represents a measure of the target concentration.
  • the analysis of the sequence modified by the deaminase treatment by hybridization-based detection methods is preferably carried out by hybridization of DNA probes, PNA probes, DNA or PNA chips or probes based on other DNA analogs (see, for example, MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag, New York, 199).
  • test kits for carrying out the detection of methylated cytosines can also be used.
  • the test kits then contain the reagents needed to perform the assay described.
  • a kit contains, for example, a deaminase or a mixture of different deaminases in the form of a concentrated solution, which is used diluted in the deamination reaction.
  • the kit contains, for example, a buffered aqueous solution, also in concentrated form (eg 10X).
  • buffer substances for example, phosphate salts, tris (hydroxymethyl) -aminomethane, citrate salts, 4- (2hydroxyethyl) -l-piperazinethanesulfonic acid (HEPES) or 2- (N-morpholino) -ethanesulfonic acid (MES) and other known to those skilled buffer substances become.
  • the concentrated buffer solution may also contain other substances favoring the deaminase reaction, such as ethylenediamine tetraacetate (EDTA) or dithiothreitol (DTT).
  • EDTA ethylenediamine tetraacetate
  • DTT dithiothreitol
  • the kit may also contain concentrated solutions of excipients (for example DNA denaturing agents such as formamide or, for example, single stranded DNA Binding Protein) which are used in dilute form in the deaminase reaction.
  • excipients for example DNA denaturing agents such as formamide or, for example, single stranded DNA Binding Protein
  • the kit can be used for instance by first being to be examined DNA target solution 1-30 thermally denatured min at 90-96 0 C and then rapidly cooled to 4 0 C as possible. Subsequently, for example, to the cooled DNA target solution, the buffer substance contained in the kit, optionally the excipients and the deaminase or the mixture of deaminases.
  • the incubation of the solution is carried out at a temperature between 4 ° C and 96 0 C for a time of a few minutes to several hours, that is, until all non-methylated cytosines are deaminated.
  • the further treatment of the DNA target solution depends on the selected analysis method. If PCR is performed after deaminase treatment, prior deactivation of deaminase is recommended. In the case of a non-thermostable deaminase, the deactivation, for example by brief heating of the solution, for example, to 90-96 0 C. Other methods of deactivating enzymes known to those skilled in the art may also be used provided that they are compatible with the subsequent analysis method.
  • Whether a purification of the mixture of salts, auxiliaries, DNA and enzymes obtained after the deaminase reaction is necessary before the analysis also depends on the requirements of the method selected for the analysis of the sequence.
  • Methods such as centrifugation by Microspin columns (for example, Microcon columns Millipore, Billerica, MA, USA) or gel filtration columns (for example, NAP columns from Amersham Biosciences, Uppsala, Sweden) are used.
  • sequence analysis can be carried out by methods known to the person skilled in the art, for example enzymatic or hybridization-based methods, for example PCR, methylation-specific PCR, methylation-specific real-time PCR, hybridization of probes in solution or on surfaces (for example DNA chips, DNA arrays).
  • enzymatic or hybridization-based methods for example PCR, methylation-specific PCR, methylation-specific real-time PCR, hybridization of probes in solution or on surfaces (for example DNA chips, DNA arrays).

Abstract

The invention relates to a method for identifying 5-methyl-cytosine in DNA sequences, in addition to test kits containing the reagents that are required to carry out the described assays.

Description

Verfahren zum Nachweis methylierter CytosineMethod for detecting methylated cytosines
Die Erfindung betrifft eine Methode zum Nachweis von 5-Methyl-Cytosin in DNA-Sequenzen sowie Testkits, die diejenigen Reagenzien enthalten, die zur Durchführung der beschriebenen Assays benötigt werden.The invention relates to a method for the detection of 5-methyl-cytosine in DNA sequences and test kits containing those reagents that are needed to carry out the described assays.
Der Nachweis von 5-Methyl-Cytosin hat ein großes Anwendungsgebiet z.B. in der Human- oder Veterinärdiagnostik, im Nahrungsmittelbereich, in der Umweltanalytik, im Pflanzenschutz, in der biochemischen bzw. pharmakologischen Forschung sowie in der forensischen Medizin.The detection of 5-methyl-cytosine has a wide field of application, e.g. in human or veterinary diagnostics, in the food sector, in environmental analysis, in crop protection, in biochemical or pharmacological research as well as in forensic medicine.
Nach dem Stand der Technik erfolgt der Nachweis von 5-Methyl-Cytosin z.B. durch Bestimmung der Länge spezifischer Restriktionsfragmente in einem Fragmentgemisch (E.M. Southern, J. Mol. Biol. 1975, 98, 503-517). Die dabei genutzten Restriktionsendonukleasen können die Basen Cytosin und 5-Methyl-Cytosin innerhalb ihrer Erkennungssequenz unterscheiden. Diese Methode ist aufwendig (Analyse durch Elektrophorese o.a.) und nicht geeignet, lange Sequenzabschnitte mit häufigen Methylierungen zu analysieren. Zudem können mit Hilfe dieses Verfahrens nur solche methylierten Cytosine analysiert werden, die sich innerhalb der Erkennungsregion einer Restriktionsendonuklease befinden. Die selektive Basenkonversion durch Behandlung mit Natriumbisulfit (R. Shapiro et al., J. Am. Chem. Soc. 1970, 92:2, 422-424) ist heute die gängigste Methode zur Analyse methylierter DNA. Grundlage dieses Verfahrens ist die effiziente und spezifische Umwandlung von Cytosin in Uracil, die durch Natriumbisulfit katalysiert wird. Da unter den gewählten Reaktionsbedingungen 5-Methyl-Cytosin nicht deaminiert wird, kann im Falle bekannter Ausgangssequenzen durch nachfolgende Analyseverfahren zwischen methylierten und nicht methylierten Cytosinen unterschieden werden. Als Analyseverfahren werden dem Fachmann bekannte Verfahren wie die Sequenzierung, Hybridisierung immobilisierter oder in Lösung befindlicher Sonden, oder die Methylierungs-spezifische PCR (M. Frommer et al., Proc. Natl. Acad. Sei., 1992, 89, 1827-1831) verwendet. Generelles Problem aller auf der Behandlung mit Natriumbisulfit beruhender Verfahren ist die signifikante Degradation der DNA (C. Grünau et al., Nucl. Acids Res., 2001, 29:13, e65). Diese Degradation des Analyten senkt die Nachweis¬ empfindlichkeit bei der Detektion von Methylierungsmustern und kann die Resultate erheblich verfälschen. Durch das kommerziell erhältliche „EZ DNA Methylation Kit™" der Firma Zymogen, Orange, USA wird die Degradation der DNA vermindert, beträgt aber immer noch >50%. Weitere Nachteile dieser Methode sind häufige Strangbrüche in der zu analysierenden DNA sowie die Schwierigkeit, ein auf Chromatographieschritten basierendes Verfahren zu automatisieren.In the prior art, the detection of 5-methyl-cytosine is e.g. by determining the length of specific restriction fragments in a fragment mixture (E.M. Southern, J. Mol. Biol. 1975, 98, 503-517). The restriction endonucleases used here can distinguish the bases cytosine and 5-methyl-cytosine within their recognition sequence. This method is expensive (analysis by electrophoresis or similar) and not suitable for analyzing long sequence sections with frequent methylations. In addition, only methylated cytosines that are within the recognition region of a restriction endonuclease can be analyzed using this method. Selective base conversion by treatment with sodium bisulfite (Shapiro, R., et al., J. Am. Chem. Soc. 1970, 92: 2, 422-424) is today the most common method for analyzing methylated DNA. The basis of this process is the efficient and specific conversion of cytosine into uracil, which is catalyzed by sodium bisulfite. Since 5-methyl-cytosine is not deaminated under the chosen reaction conditions, it can be distinguished in the case of known starting sequences by subsequent analysis methods between methylated and non-methylated cytosines. The methods of analysis are methods known to the person skilled in the art, such as sequencing, hybridization of immobilized or solution-containing probes, or methylation-specific PCR (M. Frommer et al., Proc. Natl. Acad. Sei., 1992, 89, 1827-1831). used. The general problem of all methods based on the treatment with sodium bisulfite is the significant degradation of the DNA (C. Grünau et al., Nucl. Acids Res., 2001, 29:13, e65). This degradation of the analyte reduces the detection sensitivity in the detection of methylation patterns and can significantly falsify the results. The commercially available "EZ DNA Methylation Kit ™" from Zymogen, Orange, USA, reduces the DNA degradation, but still> 50%.) Other disadvantages of this method include frequent strand breaks in the DNA to be analyzed, as well as the difficulty automate chromatography based on chromatographic steps.
Die Enzymfamilie der Cytidin-Deaminasen ist seit langem bekannt. Erst in 2002/2003 wurden jedoch Cytidin-Deaminasen beschrieben, die nicht nur die Deaminierung der Nucleobase, des Nucleosids oder des Nucleotids katalysieren, sondern auch die Deaminierung sequenzinterner Cytosine in DNA- Einzelsträngen katalysieren (R. Bransteitter et al., Proc. Natl. Acad. Sei. USA, 2003, 100:7, 4102- 4107; P. Pham et al., Nature, 2003, 424, 103-107). Für z.B. Aktivierungs-iαduzierte Cytidin Deaminase (AID) wurde auch die Unterscheidung zwischen Cytosin und 5-Methyl-Cytosin beschrieben (R. Bransteitter et al., Proc. Natl. Acad. Sei. USA, 2003, 100:7, 4102-4107). In WO 98/45472 wird eine Methode zum Screening von Inhibitoren der Cytidin-Deaminase beschrieben. Die Anmeldung beschreibt die Verwendung kurzer, synthetischer RNA-Sequenzen, die mit Cytidin- Deaminase in Gegenwart der potenziellen Inhibitioren inkubiert werden. Durch Primer-vermittelte Single Base Extension wurde geprüft, ob eine Konversion C->U erfolgt war, oder ob die Deaminase- Aktivität inhibiert wurde.The enzyme family of cytidine deaminases has long been known. However, it was not until 2002/2003 that cytidine deaminases were described, which not only deaminate the nucleobase, the nucleoside or the nucleotide, but also catalyze the deamination of sequence-internal cytosines into DNA single strands (Bransteitter, R., et al., Proc. Natl. Acad., See, USA, 2003, 100: 7, 4102-4107, P. Pham et al. , Nature, 2003, 424, 103-107). For eg activating-induced cytidine deaminase (AID), the distinction between cytosine and 5-methyl-cytosine has also been described (R. Bransteitter et al., Proc. Natl. Acad. Sci., USA, 2003, 100: 7, 4102-4107 ). In WO 98/45472 a method for the screening of inhibitors of cytidine deaminase is described. The application describes the use of short, synthetic RNA sequences which are incubated with cytidine deaminase in the presence of the potential inhibitors. Primer-mediated single base extension was used to assess whether conversion C-> U had occurred or whether deaminase activity was inhibited.
Alle diese Verfahren zum Nachweis methylierter Cytosine gemäß Stand der Technik haben den Nachteil, dass die Target-DNA zumindest teilweise der Degradation unterliegt. Zudem sind die zur Deaminierung eingesetzten Chemikalien häufig kanzerogen.All of these methods for detecting methylated cytosines according to the prior art have the disadvantage that the target DNA is at least partially subject to degradation. In addition, the chemicals used for deamination are often carcinogenic.
Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde, ein Verfahren zur schonenden, spezifischen Konversion nichtmethylierter Cytosine in Uracil bereitzustellen. Idealerweise kann es in Kombination mit dem Fachmann bekannten Nachweisverfahren wie z.B. PCR, Sequenzierung, Hybridisierung in Lösung oder auf Arrays zur Analyse von Methylierungsmustern verwendet werden.The present invention is therefore based on the object to provide a method for gentle, specific conversion of non-methylated cytosines in uracil. Ideally, it may be used in combination with detection methods known to those skilled in the art such as e.g. PCR, sequencing, hybridization in solution or on arrays for analysis of methylation patterns.
Die erfindungsgemäße Lösung der beschriebenen Aufgabe besteht in der Substitution der Bisulfϊt- Behandlung durch eine spezifische, enzymatisch katalysierte Deaminierung der nicht methyliertenThe solution according to the invention of the described object consists in the substitution of the Bisulfϊt treatment by a specific, enzymatically catalyzed deamination of the unmethylated
Cytosine. Für die enzymatische Behandlung wird erfindungsgemäß eine Deaminase, bevorzugt eineCytosine. For the enzymatic treatment according to the invention a deaminase, preferably a
Cytidin-Deaminase, besonders bevorzugt eine Cytidin-Deaminase ausgewählt aus der Gruppe derCytidine deaminase, particularly preferably a cytidine deaminase selected from the group of
APOBEC-Enzyme, ganz besonders bevorzugt AID oder APOBECl, verwendet. Erfindungsgemäß kann zur Katalyse der Deaminierung auch eine Deaminase verwendet werden, die durch biotechnologische Verfahren, wie sie dem Fachmann bekannt sind, durch Veränderung einer natürlich vorkommenden Deaminase, hergestellt wurde. Die Isolierung der natürlich vorkommendenAPOBEC enzymes, most preferably AID or APOBECl. According to the invention, a deaminase which has been prepared by biotechnological methods, as known to the person skilled in the art, by altering a naturally occurring deaminase, can also be used for catalyzing the deamination. The isolation of the naturally occurring
Deaminase erfolgt durch dem Fachmann bekannte Verfahren und ist zum Beispiel beschrieben in R.Deaminase is carried out by methods known to those skilled in the art and is described, for example, in R.
Bransteitter et al., Proc. Natl. Acad. Sei. USA, 2003 100: 7, 4102-4107, in S. K. Dickerson et al., J.Bransteitter et al., Proc. Natl. Acad. Be. USA, 2003 100: 7, 4102-4107, in S.K. Dickerson et al., J.
Exp. Med., 2003, 197, 10, 1291-1296 und in S. K. Petersen-Mahrt, M. S. Neuberger, J.Biol. Chem. 2003, 278, 22, 19583-19586.Exp. Med., 2003, 197, 10, 1291-1296 and in S.K. Petersen-Mahrt, M.S. Neuberger, J.Biol. Chem. 2003, 278, 22, 19583-19586.
Dementsprechend sind Verfahren zum Nachweis von methylierten Cytosinen, enthaltend die Schritte A) Bereitstellen eines Enzyms, das selektiv die Deaminierung nichtmethylierter Cytidine zu Uracil katalysiert.Accordingly, methods for the detection of methylated cytosines containing the steps A) Providing an enzyme that selectively catalyzes the deamination of non-methylated cytidine to uracil.
B) Inkubation einer Target-Nukleinsäure mit dem Enzym unter Konvertierung nichtmethy¬ lierter Cytosine in UracilB) incubation of a target nucleic acid with the enzyme with conversion of non-methylated cytosines into uracil
C) Analyse der veränderten Target-NukleinsäureC) Analysis of the modified target nucleic acid
Gegenstand der vorliegenden Erfindung.Subject of the present invention.
Erfmdungsgemäß kann auch ein Gemisch verschiedener Deaminasen für den Deaminierungsschritt A) verwendet werden. Hierdurch können Unterschiede in der Substratspezifität verschiedener Deaminasen ausgeglichen werden.According to the invention, a mixture of different deaminases can also be used for the deamination step A). As a result, differences in the substrate specificity of various deaminases can be compensated.
Die Target-DNA wird erfindungsgemäß mit dem Enzym / den Enzymen unter solchen Bedingungen inkubiert, unter denen die zu deaminierende DNA einzelsträngig vorliegt. Dieses kann z.B. durch thermische Denaturierung der DNA (z.B. bei 90-950C) und anschließendes schnelles Herunterkühlen unter weitgehendem Erhalt der einzelsträngigen Form (z.B. bei 40C) erfolgen (siehe zum Beispiel: M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag, New York, 1998, S. 9 ff.).According to the invention, the target DNA is incubated with the enzyme (s) under conditions in which the DNA to be deaminated is single-stranded. This can be done, for example, by thermal denaturation of the DNA (eg at 90-95 0 C) and then rapid cooling down to substantially maintain the single-stranded form (eg at 4 0 C) (see, for example: MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag , New York, 1998, p. 9 ff.).
Alternativ kann die enzymatische Deaminierung auch unter Bedingungen durchgeführt werden, unter denen zwar die DNA, nicht aber das Enzym denaturiert vorliegt. Durch niedrige Ionenkonzentration, insbesondere durch niedrige Kationenkonzentration und durch Verdünnung der DNA-Target-Lösung kann ebenfalls die Reassoziation denaturierter DNA verlangsamt werden. Wird in die DNA-Target- Lösung Formamid gegeben, so wird ebenfalls der Schmelzpunkt der DNA gesenkt und somit die Reassoziation verlangsamt. Auch durch Einsatz von Proteinen, die an Einzelstrang-DNA binden, kann die DNA in ihrer einzelsträngigen Form erhalten werden. Ein Beispiel für ein solches DNA- bindendes Protein ist das "single stranded DNA Binding Protein", das zum Beispiel von der Firma Promega, Madison, WI, USA, vertrieben wird. Die oben genannten Methoden können einzeln oder in geeigneten Kombinationen miteinander eingesetzt werden, um das DNA-Substrat für die Deaminase- Reaktion zugänglich zu machen.Alternatively, the enzymatic deamination can also be carried out under conditions under which the DNA, but not the enzyme, is denatured. Low ion concentration, in particular low cation concentration and dilution of the DNA target solution can also slow the reassociation of denatured DNA. If formamide is added to the DNA target solution, the melting point of the DNA is also lowered, slowing down reassociation. Also, by using proteins that bind to single-stranded DNA, the DNA can be obtained in its single-stranded form. An example of such a DNA-binding protein is the single-stranded DNA binding protein sold, for example, by Promega, Madison, WI, USA. The above-mentioned methods can be used individually or in suitable combinations with one another in order to make the DNA substrate available for the deaminase reaction.
Durch die Behandlung der Target-DNA mit Cytidin-Deaminase werden alle nicht methylierten Cytosine in Uracil umgewandelt. Dieser Schritt erfolgt erfϊndungsgemäß durch Inkubation der Deaminase oder des Gemisches der Deaminasen mit dem DNA-Target unter jeweils optimierten Temperatur- und Pufferbedingungen, die die Einzelsträngigkeit des Targets bei gleichzeitig optimalem Umsatz des Enzyms / Enzymgemisches gewährleisten. - A -By treating the target DNA with cytidine deaminase, all unmethylated cytosines are converted to uracil. According to the invention, this step is carried out by incubating the deaminase or the mixture of deaminases with the DNA target under respectively optimized temperature and buffer conditions which ensure the single-strandedness of the target while optimally converting the enzyme / enzyme mixture. - A -
Anschließend wird die DNA-Sequenz analysiert, wobei die Umwandlung einer Cytosinposition in Uracil anzeigt, dass an dieser Sequenzposition keine Methylierung vorlag, während unverändert gebliebene Cytosinpositionen eine Methylierung trugen. Auf diese Weise kann das Methylierungs- muster der DNA analysiert werden.Subsequently, the DNA sequence is analyzed, wherein the conversion of a cytosine position into uracil indicates that there was no methylation at this sequence position, while unchanged cytosine positions carried methylation. In this way, the methylation pattern of the DNA can be analyzed.
Die Analyse der DNA-Sequenz erfolgt zum Beispiel durch dem Fachmann bekannte Hybridisierungs- basierte oder enzymatische Nachweisverfahren. Die Analyse der durch die Deaminase-Behandlung veränderten Sequenz durch enzymatische Verfahren wird bevorzugt durch eine PCR-Reaktion erfolgen, die zum Beispiel als methylierungs-spezifische PCR (für eine Übersicht siehe zum Beispiel: C. Dahl, Biogerontology 2003, 4, 4, 233-250) oder methylierungs-spezifische real-time PCR (Beispiel: Y.M. Lo et al., Cancer Research 1999, 59, 3899-3903) ausgeführt wird.The analysis of the DNA sequence is carried out, for example, by hybridization-based or enzymatic detection methods known to the person skilled in the art. The analysis of the sequence modified by the deaminase treatment by enzymatic methods is preferably carried out by a PCR reaction, for example as a methylation-specific PCR (for a review see, for example: C. Dahl, Biogerontology 2003, 4, 4, 233 -250) or methylation-specific real-time PCR (example: YM Lo et al., Cancer Research 1999, 59, 3899-3903).
Ebenfalls bevorzugt ist die Durchführung einer Primer-Verlängerung oder einer Sequenzierreaktion, besonders bevorzugt nach dem Sanger-Verfahren (für eine Übersicht siehe zum Beispiel: L.T. Franca et al., Q. Rev. Biophys. 2002, 35, 2, 169-200). Die Sequenzierung bietet insbesondere dann Vorteile, wenn das Methylierungsmuster der zu analysierenden Sequenz unbekannt ist.It is likewise preferred to carry out a primer extension or a sequencing reaction, particularly preferably according to the Sanger method (for an overview see, for example: LT Franca et al., Q. Rev. Biophys., 2002, 35, 2, 169-200) , Sequencing offers particular advantages if the methylation pattern of the sequence to be analyzed is unknown.
Wenn die Analyse einer bekannten Sequenz mit bekanntem Methylierungsmuster nach erfolgter Deaminase-Behandlung durch methylierungs-spezifische real-time PCR erfolgt, so wird die veränderte Nukleinsäure mit zwei Sätzen von Primern und zugehörigen real-time Probes amplifiziert, wobei der eine Satz beispielsweise nur dann bindet, wenn alle Cytosine unmethyliert sind und der zweite Satz beispielsweise nur dann bindet, wenn die Sequenz an bekannten Positionen methyliert ist. Die zwei verschiedenen real-time Probes können dabei durch zwei verschiedene Fluorophore (z.B. Fluorescein und Rhodamin) unterschieden werden. Während der real-time PCR wird die Zunahme der Fluoreszenz in Folge der Target-Amplifϊkation bestimmt. Die Anzahl der PCR-Zyklen, die zur Überschreitung eines festgelegten Schwellwertes der Fluoreszenz benötigt wird, definiert man als sogenannten Ct-Wert (Cycle threshold). Der Ct-Wert kann über eine Eichkurve in Beziehung zur eingesetzten Target-Konzentration gesetzt werden und stellt somit ein Maß für die Target- Konzentration dar. Das Verhältnis der Ct- Werte der mit den beiden Sätzen an Primern und Sonden durchgeführten real-time PCR-Reaktionen zueinander ergibt nach entsprechender Normierung auf die Eichkurve den Methylierungsgrad der untersuchten Probe.If the analysis of a known sequence with known methylation pattern after deaminase treatment by methylation-specific real-time PCR, so the modified nucleic acid is amplified with two sets of primers and associated real-time probes, the one set, for example, only binds if all cytosines are unmethylated and the second set binds, for example, only if the sequence is methylated at known positions. The two different real-time probes can be distinguished by two different fluorophores (e.g., fluorescein and rhodamine). During real-time PCR, the increase in fluorescence due to the target amplification is determined. The number of PCR cycles that is needed to exceed a specified threshold of fluorescence is defined as the so-called Ct value (Cycle threshold). The Ct value can be set via a calibration curve in relation to the target concentration used and thus represents a measure of the target concentration. The ratio of the Ct values of the real-time PCR reaction performed with the two sets of primers and probes. Reactions to each other, after appropriate standardization on the calibration curve, gives the degree of methylation of the sample examined.
Die Analyse von Methylierungsmustern nach der Natriumbisulfϊt-Methode mit Hilfe der real-time PCR ist beispielsweise beschrieben in Y.M. Lo et al., Cancer Research 1999, 59, 3899-3903.Analysis of methylation patterns by the sodium bisulfite method using real-time PCR is described, for example, in Y.M. Lo et al., Cancer Research 1999, 59, 3899-3903.
Die Analyse der durch die Deaminase-Behandlung veränderten Sequenz durch Hybridisierungs- basierte Nachweisverfahren erfolgt bevorzugt durch Hybridisierung von DNA-Sonden, PNA-Sonden, DNA oder PNA Chips oder Sonden auf der Basis anderer DNA-Analoga (siehe zum Beispiel M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag, New York, 199).The analysis of the sequence modified by the deaminase treatment by hybridization-based detection methods is preferably carried out by hybridization of DNA probes, PNA probes, DNA or PNA chips or probes based on other DNA analogs (see, for example, MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag, New York, 199).
Besonders bevorzugt ist die Analyse der durch die Deaminase-Behandlung veränderten Sequenz an DNA- oder PNA-Chips. Solche Verfahren sind dem Fachmann bekannt, die Analyse von Methylierungsmustern auf DNA-Chips nach der Natriumbisulfit-Methode ist beispielsweise beschrieben in H. Shi et al, Journal of Cellular Biochemistry 2003, 88, 138-143.Particularly preferred is the analysis of the sequence modified by deaminase treatment on DNA or PNA chips. Such methods are known to those skilled in the art, the analysis of methylation patterns on DNA chips according to the sodium bisulfite method is described for example in H. Shi et al, Journal of Cellular Biochemistry 2003, 88, 138-143.
Erfindungsgemäß können auch Testkits zur Durchführung des Nachweises methylierter Cytosine eingesetzt werden. Die Testkits enthalten dann diejenigen Reagenzien, die zur Durchführung des beschriebenen Assays benötigt werden. Ein solches Kit enthält beispielsweise eine Deaminase oder ein Gemisch verschiedener Deaminasen in Form einer konzentrierten Lösung, die verdünnt in die Deaminierungsreaktion eingesetzt wird. Außerdem enthält das Kit beispielsweise eine gepufferte wässrige Lösung, ebenfalls in konzentrierter Form (z.B. 1Ox). Als Puffersubstanzen können z.B. Phosphatsalze, Tris-(hydroxymethyl)-aminomethan, Citratsalze, 4-(2hydroxyethyl)-l-piperazinethan- sulfonsäure (HEPES) oder 2-(N-Morpholino)-ethansulfonsäure (MES) sowie andere dem Fachmann bekannte Puffersubstanzen eingesetzt werden. Die konzentrierte Pufferlösung kann auch andere, die Deaminase-Reaktion begünstigende Stoffe wie zum Beispiel Ethylendiamintetraacetat (EDTA) oder Dithiothreitol (DTT) enthalten. Das Kit kann auch konzentrierte Lösungen von Hilfsstoffen (zum Beispiel von DNA-denaturierenden Agenzien wie Formamid oder zum Beispiel single stranded DNA Binding Protein) enthalten, die in verdünnter Form in die Deaminase-Reaktion eingesetzt werden. Das Kit kann beispielsweise eingesetzt werden, indem zunächst die zu untersuchende DNA-Target Lösung 1-30 min bei 90-960C thermisch denaturiert und anschließend möglichst schnell auf 40C abgekühlt wird. Anschließend gibt man zum Beispiel zu der gekühlten DNA-Target-Lösung die im Kit enthaltene Puffersubstanz, ggf. die Hilfsstoffe sowie die Deaminase oder das Gemisch der Deaminasen. Anschließend erfolgt die Inkubation der Lösung bei einer Temperatur zwischen 4°C und 960C für eine Zeit von wenigen Minuten bis zu mehreren Stunden, das heißt solange, bis alle nichtmethylierten Cytosine desaminiert sind. Nach erfolgter Deaminase-Reaktion richtet sich die weitere Behandlung der DNA-Target-Lösung nach der jeweils gewählten Analysemethode. Wird nach der Deaminase-Behandlung eine PCR durchgeführt, ist die vorherige Desaktivierung der Deaminase empfehlenswert. Im Falle einer nicht thermostabilen Deaminase kann die Desaktivierung z.B. durch kurzes Erhitzen der Lösung z.B. auf 90-960C erfolgen. Andere, dem Fachmann bekannte Methoden zur Desaktivierung von Enzymen können ebenfalls eingesetzt werden, sofern sie mit der anschließenden Analysemethode kompatibel sind. Ob vor der Analyse eine Aufreinigung des nach der Deaminase-Reaktion erhaltenen Gemisches aus Salzen, Hilfsstoffen, DNA und Enzymen notwendig ist, richtet sich ebenfalls nach den Anforderungen der zur Seqύenzanalyse ausgewählten Methode. Zur Aufreinigung und Entsalzung des Gemisches können dem Fachmann bekannte Methoden wie z.B. die Zentrifugation durch Microspin-Säulen (beispielsweise Microcon-Säulen der Firma Millipore, Billerica, MA, USA) oder Gelfiltrationssäulen (beispielsweise NAP-Säulen der Firma Amersham Biosciences, Uppsala, Schweden) eingesetzt werden. Die Sequenzanalyse kann durch dem Fachmann bekannte Verfahren wie zum Beispiel enzymatische oder hybridisierungs- basierte Verfahren, zum Beispiel PCR, methylierungs-spezifische PCR, methylierungs-spezifische real-time PCR, Hybridisierung von Sonden in Lösung oder an Oberflächen (zum Beispiel DNA- Chips, DNA-Arrays) erfolgen.According to the invention, test kits for carrying out the detection of methylated cytosines can also be used. The test kits then contain the reagents needed to perform the assay described. Such a kit contains, for example, a deaminase or a mixture of different deaminases in the form of a concentrated solution, which is used diluted in the deamination reaction. In addition, the kit contains, for example, a buffered aqueous solution, also in concentrated form (eg 10X). As buffer substances, for example, phosphate salts, tris (hydroxymethyl) -aminomethane, citrate salts, 4- (2hydroxyethyl) -l-piperazinethanesulfonic acid (HEPES) or 2- (N-morpholino) -ethanesulfonic acid (MES) and other known to those skilled buffer substances become. The concentrated buffer solution may also contain other substances favoring the deaminase reaction, such as ethylenediamine tetraacetate (EDTA) or dithiothreitol (DTT). The kit may also contain concentrated solutions of excipients (for example DNA denaturing agents such as formamide or, for example, single stranded DNA Binding Protein) which are used in dilute form in the deaminase reaction. The kit can be used for instance by first being to be examined DNA target solution 1-30 thermally denatured min at 90-96 0 C and then rapidly cooled to 4 0 C as possible. Subsequently, for example, to the cooled DNA target solution, the buffer substance contained in the kit, optionally the excipients and the deaminase or the mixture of deaminases. Subsequently, the incubation of the solution is carried out at a temperature between 4 ° C and 96 0 C for a time of a few minutes to several hours, that is, until all non-methylated cytosines are deaminated. After the deaminase reaction has taken place, the further treatment of the DNA target solution depends on the selected analysis method. If PCR is performed after deaminase treatment, prior deactivation of deaminase is recommended. In the case of a non-thermostable deaminase, the deactivation, for example by brief heating of the solution, for example, to 90-96 0 C. Other methods of deactivating enzymes known to those skilled in the art may also be used provided that they are compatible with the subsequent analysis method. Whether a purification of the mixture of salts, auxiliaries, DNA and enzymes obtained after the deaminase reaction is necessary before the analysis also depends on the requirements of the method selected for the analysis of the sequence. For purification and desalting of the mixture known to the expert Methods such as centrifugation by Microspin columns (for example, Microcon columns Millipore, Billerica, MA, USA) or gel filtration columns (for example, NAP columns from Amersham Biosciences, Uppsala, Sweden) are used. The sequence analysis can be carried out by methods known to the person skilled in the art, for example enzymatic or hybridization-based methods, for example PCR, methylation-specific PCR, methylation-specific real-time PCR, hybridization of probes in solution or on surfaces (for example DNA chips, DNA arrays).
Vorteil des erfindungsgemäßen Verfahrens gegenüber dem Stand der Technik ist die selektive, enzymatisch katalysierte Deaminierung unter milden Reaktionsbedingungen, unter denen keine Degradation der Target-DNA erfolgt. Ein weiterer Vorteil des Verfahrens ist, dass keine kanzerogenen Chemikalien für die Deaminierung eingesetzt werden. Advantage of the method according to the invention over the prior art is the selective, enzymatically catalyzed deamination under mild reaction conditions under which no degradation of the target DNA occurs. Another advantage of the method is that no carcinogenic chemicals are used for deamination.

Claims

Patentansprüche claims
1. Verfahren zum Nachweis von methylierten Cytosinen, mit den Schritten1. A method for detecting methylated cytosines, comprising the steps
A) Bereitstellen eines Enzyms, das selektiv die Deaminierung nichtmethylierter Cytidine zu Uracil katalysiert.A) Providing an enzyme that selectively catalyzes the deamination of non-methylated cytidine to uracil.
B) Inkubation einer Target-Nukleinsäure mit dem Enzym unter Konvertierung nichtmethylierter Cytosine in UracilB) Incubation of a target nucleic acid with the enzyme with conversion of non-methylated cytosines into uracil
C) Analyse der veränderten Target-NukleinsäureC) Analysis of the modified target nucleic acid
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass das Enzym eine Cytidin- Deaminase ist.2. The method according to claim 1, characterized in that the enzyme is a cytidine deaminase.
3. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass das Enzym aus der Gruppe der APOBEC-Enzyme ausgewählt ist.3. The method according to claim 1, characterized in that the enzyme is selected from the group of APOBEC enzymes.
4. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass das Enzym eine Aktivierungs- induzierte Cytidin-Deaminase (AID) oder APOBECl ist.4. The method according to claim 1, characterized in that the enzyme is an activation-induced cytidine deaminase (AID) or APOBECl.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass das Enzym eine Deaminase ist, die durch biotechnologische Verfahren aus einer natürlich vorkommenden Deaminase erzeugt wurde.5. The method according to any one of claims 1 to 4, characterized in that the enzyme is a deaminase, which was generated by biotechnological methods from a naturally occurring deaminase.
6. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass ein Gemisch verschiedener Enzyme für den Deaminierungsschritt Verwendung findet.6. The method according to any one of claims 1 to 5, characterized in that a mixture of different enzymes is used for the Deaminierungsschritt.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass die Target- Nukleinsäure während der Inkubation mit dem Enzym einzelsträngig vorliegt.7. The method according to any one of claims 1 to 6, characterized in that the target nucleic acid is present during the incubation with the enzyme single-stranded.
8. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass die Analyse der veränderten Target-Nukleinsäure durch enzymatische oder durch Hybridisierungs- basierte Verfahren erfolgt.8. The method according to any one of claims 1 to 7, characterized in that the analysis of the modified target nucleic acid by enzymatic or by hybridization-based method.
9. Verfahren nach einem der Ansprüche 1 bis 8, dadurch gekennzeichnet, dass die Analyse der veränderten Target-Nukleinsäure durch PCR, methylierungs-spezifische PCR, methy- lierungs-spezifische real-time PCR, Primer-Verlängerung oder eine Sequenzierreaktion erfolgt. 9. The method according to any one of claims 1 to 8, characterized in that the analysis of the modified target nucleic acid by PCR, methylation-specific PCR, methylation-specific real-time PCR, primer extension or a sequencing reaction takes place.
10. Testkit, das diejenigen Reagenzien enthält, die zur Durchführung der Assays nach einem der Ansprüche 1-10 benötigt werden.A kit containing the reagents needed to perform the assays of any one of claims 1-10.
Der Anspruch auf Hybridisierungsbasierte Systeme fehlt jetzt aus Kostengründen, kann aber im Prüfungsverfahren nachgeschoben werden, da im Text offenbart. The claim to hybridization-based systems is now lacking for cost reasons, but can be postponed in the examination process, as disclosed in the text.
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