[go: up one dir, main page]

WO2006050864A1 - Procede pour mettre en evidence de la cytosine methylee - Google Patents

Procede pour mettre en evidence de la cytosine methylee Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
deaminase
dna
nucleic acid
analysis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2005/011831
Other languages
German (de)
English (en)
Inventor
Jens Burmeister
Walter Weichel
Reinhard Koch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
Original Assignee
Bayer Technology Services GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Technology Services GmbH filed Critical Bayer Technology Services GmbH
Publication of WO2006050864A1 publication Critical patent/WO2006050864A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour mettre en évidence de la 5-méthyle-cytosine dans des séquences d'ADN, ainsi que des kits d'essais contenant les réactifs nécessaires à la réalisation des essais décrits.
PCT/EP2005/011831 2004-11-12 2005-11-04 Procede pour mettre en evidence de la cytosine methylee Ceased WO2006050864A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200410054729 DE102004054729A1 (de) 2004-11-12 2004-11-12 Verfahren zum Nachweis methylierter Cytosine
DE102004054729.7 2004-11-12

Publications (1)

Publication Number Publication Date
WO2006050864A1 true WO2006050864A1 (fr) 2006-05-18

Family

ID=35501286

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/011831 Ceased WO2006050864A1 (fr) 2004-11-12 2005-11-04 Procede pour mettre en evidence de la cytosine methylee

Country Status (2)

Country Link
DE (1) DE102004054729A1 (fr)
WO (1) WO2006050864A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015514397A (ja) * 2012-03-15 2015-05-21 ニユー・イングランド・バイオレイブス・インコーポレイテツド シトシンとこれの修飾物とを識別するための、およびメチローム分析のための方法および組成物
CN114032287A (zh) * 2021-11-24 2022-02-11 竹石生物科技(苏州)有限公司 Dna甲基化测序文库及其构建方法和检测方法
WO2024147904A1 (fr) * 2023-01-06 2024-07-11 Illumina, Inc. Réduction des uraciles par polymérase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210888B1 (en) * 1997-04-10 2001-04-03 Itzik Harosh Technique for screening inhibitors of deamination enzyme activity
DE10331107B3 (de) * 2003-07-04 2004-12-02 Epigenomics Ag Verfahren zum Nachweis von Cytosin-Methylierungen in DNA mittels Cytidin-Deaminasen

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210888B1 (en) * 1997-04-10 2001-04-03 Itzik Harosh Technique for screening inhibitors of deamination enzyme activity
DE10331107B3 (de) * 2003-07-04 2004-12-02 Epigenomics Ag Verfahren zum Nachweis von Cytosin-Methylierungen in DNA mittels Cytidin-Deaminasen
WO2005005660A1 (fr) * 2003-07-04 2005-01-20 Epigenomics Ag Methode utilisant des cytidine deaminases pour detecter des methylations de la cytosine dans l'adn

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRANSTEITTER R ET AL: "Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 100, no. 7, 1 April 2003 (2003-04-01), pages 4102 - 4107, XP002302399, ISSN: 0027-8424 *
LARIJANI MANI ET AL: "Methylation protects cytidines from AID-mediated deamination", MOLECULAR IMMUNOLOGY, vol. 42, no. 5, March 2005 (2005-03-01), pages 599 - 604, XP002361963, ISSN: 0161-5890 *
MORGAN HUGH D ET AL: "Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues - Implications for epigenetic reprogramming", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 50, 24 September 2004 (2004-09-24), pages 52353 - 52360, XP002361964, ISSN: 0021-9258 *
PHAM P ET AL: "Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation", NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 424, no. 6944, 3 July 2003 (2003-07-03), pages 103 - 107, XP002302398, ISSN: 0028-0836 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015514397A (ja) * 2012-03-15 2015-05-21 ニユー・イングランド・バイオレイブス・インコーポレイテツド シトシンとこれの修飾物とを識別するための、およびメチローム分析のための方法および組成物
CN114032287A (zh) * 2021-11-24 2022-02-11 竹石生物科技(苏州)有限公司 Dna甲基化测序文库及其构建方法和检测方法
CN114032287B (zh) * 2021-11-24 2024-09-17 竹石生物科技(苏州)有限公司 Dna甲基化测序文库及其构建方法和检测方法
WO2024147904A1 (fr) * 2023-01-06 2024-07-11 Illumina, Inc. Réduction des uraciles par polymérase

Also Published As

Publication number Publication date
DE102004054729A1 (de) 2006-05-18

Similar Documents

Publication Publication Date Title
EP0925373B1 (fr) Procede pour la detection d'acides nucleiques par determination de la masse
DE69421277T2 (de) NUKLEINSäURE-SEQUENZANALYSE DURCH DIE METHODE DER PARALLELEN PRIMEREXTENSION
DE69432919T2 (de) Verfahren für den Nachweis spezifischer Polynukleotiden
DE69233458T2 (de) Nukleinsäuretypisierung durch polymeraseverlängerung von oligonukleotiden unter verwendung von terminator-mischungen
DE69231124T2 (de) Schnelle bestimmungsverfahren für amplifikationsprodukte
DE602004009038T2 (de) Verbessertes verfahren zur behandlung durch bisulfit
DE69128077T2 (de) Nuklein-säure amplifikation mit dns abhängigen rns polymerasen aktivität von rns-replikasen
DE69003477T2 (de) Verfahren zum Nachweis von Nucleinsäuren.
DE69008179T2 (de) Bestimmung eines mutationsspektrums.
DE102007046990B4 (de) Verfahren zur Detektion kleiner Oligonukleotide
DE112011100297T5 (de) Zusammensetzungen, Verfahren und damit in Zusammenhang stehende Anwendungen zum Spalten modifizierter DNA
DE10120798A1 (de) Verfahren zur Bestimmung der Genexpression
DE69421618T2 (de) In situ extraktion mikrobieller dna
DE102016124844B3 (de) DNA-Sonden für eine In-Situ-Hybridisierung von Chromosomen
DE69633723T2 (de) Methode zur detektion von punktmutanten mittels nukleinsäurereperaturenzym und in vitro mutagenese
DE69421578T2 (de) Oligonukleotid-Bestimmung
EP3350340B1 (fr) Test de vérification de produits d'amplification primaires d'acides nucléiques dans un processus réactionnel continu et analyse directe au moyen d'un procédé immunochromatographique
EP2126134A1 (fr) Procede et kit de test permettant la detection rapide de sequences d'acides nucleiques specifiques, notamment la detection de mutations ou de polymorphismes d'un seul nucleotide (snp)
DE3910151A1 (de) 5'-bromo-2'-desoxyuridinmarkierte dna- oder rna- sonden
WO2017046192A1 (fr) Test de vérification de produits d'amplification primaires d'acides nucléiques au cours d'un processus réactionnel continu et analyse directe au moyen d'un procédé électrophorétique
DE102006014879B4 (de) RNA-Markierungsverfahren
EP1364068B1 (fr) Procede et kit permettant l'identification d'adn specifique des especes animales dans un echantillon
WO2006050864A1 (fr) Procede pour mettre en evidence de la cytosine methylee
DE60129220T2 (de) Detektion und charakterisierung der aktivität von proteinen, welche an der reparatur von dna-läsionen beteiligt sind
DE60201441T2 (de) Verfahren zur analyse der dna-methylierung unter verwendung von fluoreszenzpolarisation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase

Ref document number: 05807820

Country of ref document: EP

Kind code of ref document: A1