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WO2003023065A1 - Types de methylation de l'adn - Google Patents

Types de methylation de l'adn Download PDF

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Publication number
WO2003023065A1
WO2003023065A1 PCT/US2002/028529 US0228529W WO03023065A1 WO 2003023065 A1 WO2003023065 A1 WO 2003023065A1 US 0228529 W US0228529 W US 0228529W WO 03023065 A1 WO03023065 A1 WO 03023065A1
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WIPO (PCT)
Prior art keywords
dna
endonuclease
recognition sequence
genomic
methylcytosine
Prior art date
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Ceased
Application number
PCT/US2002/028529
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English (en)
Inventor
Xun Wang
Hur-Song Chang
Jerzy Paszkowski
Liang Shi
Tong Zhu
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Syngenta Participations AG
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Syngenta Participations AG
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Publication of WO2003023065A1 publication Critical patent/WO2003023065A1/fr
Anticipated expiration legal-status Critical
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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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to genomics and in particular to a method for the rapid assessment of the genome wide distribution of DNA methylation using restriction endonucleases which are sensitive to methylation within their recognition site in combination with size fractionation of the restricted DNA and hybridization to a DNA chip.
  • DNA methylation is a ubiquitous biological process that occurs in diverse organisms ranging from bacteria to humans. During this process, DNA methyltransferases catalyze primarily the post-replicative addition of a methyl group to the N6 position of adenine or the C5 position of cytosine. In higher eukaryotes, DNA methylation plays a central role in epigenetic regulation of gene expression and in particular in transcriptional gene silencing, genomic imprinting and embryonic development. Aberrations in DNA methylation have been implicated in aging and various diseases including cancer.
  • a method for determining the methylation state within a genomic sequence context based on the use of restriction endonucleases which require the recognition sequence to be unmethylated to allow cleavage at the site has been described by Bird et al, J. Mol. Biol. 118, 27- 47, 1978.
  • the fragments resulting from cleavage of unmethylated recognition sites are detected by gel electrophoresis, transferred to a membrane and hybridized to a labeled probe corresponding to the DNA fragment to be examined.
  • the resulting hybridization pattern reflects the methylation pattern of the DNA.
  • the sensitivity of this method was increased in a variant combined with PCR (Shemer, R. et al., Proc. Natl. Acad. Sci USA, 93, 6371-6376, 1996).
  • Amplification by two primers located on both sides of the recognition sequence only occurs after cleavage if the recognition sequence is in the methylated form. With both variants only methylation of individual positions is examined.
  • An array is an orderly arrangement of DNA samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns.
  • An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample, h general, arrays are described as macroarrays or microarrays, the difference being the size of the sample spots.
  • Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contain thousands of spots.
  • DNA microarray or DNA chips are fabricated by robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers infqrmation on thousands of genes simultaneously.
  • a "probe” is frequently considered to be a tethered nucleic acid with known sequence, whereas a “target” is a free nucleic acid sample whose identity or abundance is analyzed.
  • microarray technology There are two major application forms for the microarray technology: (1) identification of a nucleotide sequence such as a gene or gene mutation and (2) determination of the expression level or abundance of a nucleotide sequence.
  • identification of a nucleotide sequence such as a gene or gene mutation
  • determination of the expression level or abundance of a nucleotide sequence There are also two variants of the DNA microarray technology, in terms of the property of arrayed DNA sequence with known identity: In variant 1, probe DNAs of 500-5,000 bases length such as cDNA are immobilized to a solid surface such as glass using robotic spotting and exposed to a set of targets either separately or in a mixture.
  • an array of oligonucleotides (20-80-mer oligos) or peptide nucleic acid (PNA) probes are synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity or abundance of complementary sequences is determined.
  • This method "historically” called DNA chips, is sold under the GeneChip® trademark.
  • GeneChip® arrays use multiple probes (e.g. 16 probe pairs for one gene in the case of the Arabidopsis chip) to interrogate a chromosomal region.
  • a GeneChip® expression array can contain probes corresponding to a number of reference and control genes. Using reference standards, it is possible to normalize data from different experiments and compare multiple experiments on a quantitative level.
  • Probe arrays are manufactured using technology that combines photolithographic methods and combinatorial chemistry. Tens to hundreds of thousands of different ohgonucleotide probes are synthesized on each array. Each probe type is located in a specific area on the probe array called a probe cell. Each probe cell contains millions of copies of a given probe. Probe arrays are manufactured in a series of cycles. A glass substrate is coated with linkers containing photo labile protecting groups. Then, a mask is applied that exposes selected portions of the probe array to ultraviolet light. Illumination removes the photo labile protecting groups enabling selective nucleoside phosphoramidite addition only at the previously exposed sites. Next, a different mask is applied and the cycle of illumination and chemical coupling is performed again.
  • oligonucleotide probes By repeating this cycle, a specific set of oligonucleotide probes is synthesized, with each probe type in a known location. The completed probe arrays are packaged into cartridges. Many companies are manufacturing oligonucleotide-based chips using alternative in situ synthesis or depositioning technologies.
  • the present invention combines the use of methylation-sensitive restriction enzymes, DNA size fractionation and DNA microarray technology in a way, which makes it feasible to examine the level and the distribution of DNA methylation on a genome wide scale. It also allows resolution down to a gene, gene fragment or any chosen DNA sequence, and can be subjected to quantification. Whereas all types of microarrays can be used in the context of the present invention, it is an important aspect that the probe arrays are hybridized with a selected size fraction of labeled genomic target DNA that has been restricted with a methylation sensitive endonuclease.
  • Hybridization intensities of different sources of genomic target DNA are then compared to each other and optionally to a control digestion with a methylation insensitive endonuclease to identify sequences showing different levels of methylation.
  • the intensities are indicative of the composition of the particular size fraction and therefore of the methylation status of the genomic targets.
  • a comparatively high intensity of hybridization of a probe with a low molecular weight target fraction resulting from cleavage with an endonuclease inhibited by the presence of 5-methylcytosine or N6-methyladenine in the recognition sequence reflects hypomethylation in the target fraction relative to other samples or controls.
  • a comparatively high intensity of hybridization of a probe with a low molecular weight target resulting from digestion with an endonuclease requiring the presence of 5-methylcytosine or N6- methyladenine in the recognition sequence reflects hypermethylation in the target fraction relative to other samples or controls.
  • the present invention teaches a method to detect differences of genomic methylation comprising (a) separately cleaving different samples of genomic DNA with a sequence specific endonuclease whose cleavage activity is inhibited by or requires the presence of 5- methylcytosine or N6-methyladenine in the recognition sequence;
  • step (c) separately hybridizing the labelled DNA fractions of step (b) to an array of DNA molecules representing a plurality of genomic DNA targets;
  • step (d) quantifying the differences of the hybridization intensity patterns obtained in step (c). Additionally, as a hybridization control, the method may optionally comprise
  • step (ii) labelling the same size fraction of the resulting DNA fragments as in step (b); and (iii) separately hybridizing the labelled DNA fractions of step (ii) to an identical array of DNA molecules representing a plurality of genomic DNA targets.
  • the method is particularly useful to distinguish different cell types on the basis of their methylation pattern, which can be extremely useful in the context of cancer diagnosis and treatment.
  • the method is not restricted to the analysis of mammalian genome methylation, but can also be used for methylation pattern analysis in plants, animals, insects, fungi or microbes. Thus, it can be used in plants to compare methylation patterns in the context of heterosis.
  • the DNA samples to be compared are of isogenic origin such as healthy versus tumour tissue of the same organism, parental DNA versus progeny or sibling DNA, or DNA of isogenic organisms only differing by one or more specific mutations. In general, with increasing genetic distance of the samples to be compared, interpretation of the results becomes more difficult.
  • a number of different endonucleases inhibited by the presence of 5-methylcytosine or N6-methyladenine can be used in the context of the present invention. Their respective recognition sequences are usually defined by 4 to 8 base pairs, shorter recognition sequences of 4 to 6 base pairs being preferred.
  • a number of particularly useful endonucleases are listed in Table 1. Endonucleases which are useful at sites of overlapping CG are shown in Table 2. Particularly preferred examples of recogmtion sequences are ACGT, GCGC, CCGG, TCGA or CGCG.
  • DNA label is only important in the sense that it needs to be compatible with the hybridization technology required for the probes immobilized on the solid array supports.
  • any method which can label DNA quantitatively can be used for this application, including but not limited to random ohgonucleotide priming , nick translation, chemical labelling of DNA such as labelling with Biotin, and light activated chemical labelling of DNA such as labelling with Psoralen-biotin and activation by UV light. Means to perform these methods are commercially available.
  • a preferred support is the Affymetrix GeneChip which can be hybridized to the target DNA according to the manufacturer's instructions.
  • the preferred size fraction to be labled primarily depends on the length of the recognition sequence cleaved by the endonuclease. Thus, for a recognition sequence of 4 base pairs the preferred fragment size is up to 3000 base pairs and preferentially between 100 and 2000 base pairs. For a 6 base pair recognition sequence a fragment size of between 100 and 6000 base pairs is suitable.
  • the specific probes of the DNA array may represent any type of genomic DNA, that is coding sequences such as cDNA or non-coding sequences such as promoters, enhancers, terminators, introns, transposons etc. In the context of methylation studies it is preferred that the specific probe arrays represent non-coding sequences such as regulatory sequences of the genome of an organism.
  • Experimental data resulting from the hybridization can be analyzed using computer software.
  • Affymetrix offers a program called GeneChip Microarray Suite. This program, for comparison between two chips, measures and normalizes the 'baseline chip' intensity values to the average signal intensity. Intensity values of the 'experimental chip' are then compared to the baseline chip and a 'difference change' is calculated.
  • the output of the software provides a qualitative call: 'increase', 'marginal increase', 'no change', 'decrease', and 'marginal decrease' as well as discrete numerical metrics used to make the call.
  • Expression elements may be ranked based on absolute level of expression or relative change in expression between a two chip comparison. All numerical data may be exported to a Microsoft Excel spreadsheet for further analysis. Expression elements are identified by a GenBank accession number and the GeneChip analysis software allows for immediate hyperlink to the GenBank entry for full sequence annotation. Examples
  • Genomic DNA of Arabidopsis thaliana mutant som8 plants (O Mittelsten Scheid et al (1998), Proc. Natl. Acad. Sci. USA 95, 632-637) and corresponding wild type plants is isolated by standard biochemical procedures ensuring high molecular weight and sufficient purity for enzymatic modifications.
  • the DNA is subjected to endonucleolytic cleavage with the sequence specific endonuclease Hpall, the activity of which is blocked by the presence of 5-methyl cytosine in the recognition sequence.
  • the same DNA sample is digested with the endonuclease Mspl.
  • endonucleolytic cleavage and agarose gel electrophoresis various size fractions are eluted from the gel and labelled using the Life Technologies DNA Labelling system with some modifications and scaled up to 200 ⁇ l:
  • a DNA size fraction eluted from the gel generally containing between 0.5-1.5 ⁇ g of DNA and preferably 0.8 ⁇ g of DNA, are mixed with lO ⁇ l H2O and 80 ⁇ l of a 2.5x Random Primer Solution and placed on ice;
  • DNA is precipitated by the addition of 22 ⁇ l 3M NaAc and 440 ⁇ l ethanol (95-99%) and leaving the DNA on dry ice for 20 minutes or at -20°C overnight;
  • the DNA is then collected by centrifugation at 4°C for 10 minutes at maximum speed of a table top centrifuge and discarding the supernatant; 7.
  • the pellet is then redissolved in 200 ⁇ l H2O and reprecipitated with ethanol (steps 5-6);
  • the commercially available GeneChip® Arabidopsis genome array used in this example contains probe sets interrogating more than 8,200 genes and more than 100 EST clusters for Arabidopsis thaliana. Eighty percent of the genes represented on the array are predicted coding sequences from genomic BAC entries. Twenty percent are from high quality cDNA sequences. The array also contains more than 100 EST clusters, sharing homology with the predicted coding sequences from BAC clones.
  • the labelled target DNA of example 1 is hybridized to the Arabidopsis Genome Array and further analyzed as described in example 1 of EP-A-999 285 and corresponding US Patent No. 6,203,989.
  • the DNA fraction which is preferentially demethylated in som8 Arabidopsis mutants, is composed of remnants of transposons and of repetitive DNA.
  • the 8000 genes studied 124 can be characterized as being related to transposable elements and the experimental data confirm that transposable elements are preferentially demethylated in som8 plants as compared to the control wild type plants.
  • the methylation level decrease of the transposons correlates well with their transcriptional reactivation in many independent examples. However, a subset of transposons, although demethylated, remains transcriptionally inactive. In addition, it is found that selected genes and members of multigene families are also subjected to demethylation and transcriptional reactivation in som8 plants similar to the subset of transposons. Among this group of genes those encoding pathogen resistance determinants are the most prominent examples.

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Abstract

La présente invention concerne une méthode qui permet de détecter des différences de méthylation génomique à l'aide de réseaux de sondes ADN représentant une pluralité de séquences génomiques hybridées sur une fraction de longueur de fragments de restriction d'ADN génomique produits par des endonucléases qui sont inhibées par la 5-méthylcytosine ou la N6-méthyladenine ou qui nécessitent ces dernières dans la séquence de reconnaissance.
PCT/US2002/028529 2001-09-06 2002-09-06 Types de methylation de l'adn Ceased WO2003023065A1 (fr)

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US7186512B2 (en) 2002-06-26 2007-03-06 Cold Spring Harbor Laboratory Methods and compositions for determining methylation profiles
US7459274B2 (en) 2004-03-02 2008-12-02 Orion Genomics Llc Differential enzymatic fragmentation by whole genome amplification
WO2009100029A1 (fr) 2008-02-01 2009-08-13 The General Hospital Corporation Utilisation de microvésicules dans le diagnostic, le pronostic et le traitement de maladies et d’affections médicales
WO2011009104A1 (fr) 2009-07-16 2011-01-20 The General Hospital Corporation Analyse d'acides nucléiques
US7901880B2 (en) 2003-10-21 2011-03-08 Orion Genomics Llc Differential enzymatic fragmentation
WO2011031877A1 (fr) 2009-09-09 2011-03-17 The General Hospital Corporation Utilisation de microvésicules dans l'analyse de profils d'acide nucléique
WO2012031008A2 (fr) 2010-08-31 2012-03-08 The General Hospital Corporation Matières biologiques liées au cancer dans des microvésicules
WO2012155014A1 (fr) 2011-05-11 2012-11-15 Exosome Diagnostics, Inc. Extraction d'acide nucléiques à partir de matériaux biologiques hétérogènes
WO2013028788A1 (fr) 2011-08-22 2013-02-28 Exosome Diagnostics, Inc. Marqueurs biologiques d'urine
WO2014055775A1 (fr) 2012-10-03 2014-04-10 Exosome Diagnostics, Inc. Utilisation de microvésicules dans le diagnostic, le pronostic et le traitement de maladies et d'états médicaux
US9243233B2 (en) 2012-07-05 2016-01-26 Thermo Fisher Scientific UAB Restriction endonucleases and their uses
WO2017181183A1 (fr) 2016-04-15 2017-10-19 Exosome Diagnostics, Inc. Détection par plasma d'acides nucléiques de kinase de lymphome anaplasique (alk) et de transcrits de fusion d'alk et leurs utilisations dans le diagnostic et le traitement du cancer
WO2017197399A1 (fr) 2016-05-13 2017-11-16 Exosome Diagnostics, Inc. Procédés automatisés et manuels pour l'isolement de vésicules extracellulaires et l'isolement conjoint d'adn acellulaire à partir de bioliquides
US9828640B2 (en) 2006-03-31 2017-11-28 Affymetrix, Inc. Analysis of methylation using nucleic acid arrays
WO2018076018A1 (fr) 2016-10-21 2018-04-26 Exosome Diagnostics, Inc. Séquençage et analyse d'acides nucléiques associés à des exosomes
WO2018102162A1 (fr) 2016-11-30 2018-06-07 Exosome Diagnostics, Inc. Méthodes et compositions pour détecter des mutations dans du plasma à l'aide d'arn exosomal et d'adn acellulaire en provenance de patients atteints d'un cancer du poumon non à petites cellules
WO2018126278A2 (fr) 2017-01-02 2018-07-05 Exosome Diagnostics, Inc. Procédés de distinction d'arn et d'adn dans une préparation combinée
US10174361B2 (en) 2011-11-10 2019-01-08 Exosome Diagnostics, Inc. Cerebrospinal fluid assay
US10301681B2 (en) 2013-08-06 2019-05-28 Exosome Diagnostics, Inc. Methods of treating a subject with a high gleason score prostate cancer
US10407717B2 (en) 2001-11-19 2019-09-10 Affymetrix, Inc. Methods of analysis of methylation
WO2020106853A1 (fr) 2018-11-20 2020-05-28 Exosome Diagnostics, Inc. Compositions et procédés de témoins internes d'isolements de microvésicules
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EP3865590A1 (fr) 2009-09-09 2021-08-18 The General Hospital Corporation Utilisation de microvésicules dans l'analyse de mutations kras
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US11899024B2 (en) 2017-07-12 2024-02-13 Exosome Diagnostics, Inc. Treatment and diagnosis of parkinson's disease using isolated and enriched populations of biofluid-derived extracellular vesicles

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US10822642B2 (en) 2001-11-19 2020-11-03 Affymetrix, Inc. Methods of analysis of methylation
US10407717B2 (en) 2001-11-19 2019-09-10 Affymetrix, Inc. Methods of analysis of methylation
US7186512B2 (en) 2002-06-26 2007-03-06 Cold Spring Harbor Laboratory Methods and compositions for determining methylation profiles
US8273528B2 (en) 2002-06-26 2012-09-25 Cold Spring Harbor Laboratory Methods and compositions for determining methylation profiles
US8361719B2 (en) 2003-10-21 2013-01-29 Orion Genomics Llc Methods for quantitative determination of methylation density in a DNA locus
US7901880B2 (en) 2003-10-21 2011-03-08 Orion Genomics Llc Differential enzymatic fragmentation
US7910296B2 (en) 2003-10-21 2011-03-22 Orion Genomics Llc Methods for quantitative determination of methylation density in a DNA locus
US8163485B2 (en) 2003-10-21 2012-04-24 Orion Genomics, Llc Differential enzymatic fragmentation
GB2422902A (en) * 2003-11-09 2006-08-09 Epigenomics Ag Method for investigating cytosine methylation in DNA sequences by means of hemimethylation sensitive restriction enzymes
WO2005045069A3 (fr) * 2003-11-09 2005-07-21 Epigenomics Ag Procede de controle de la methylation de la cytosine dans des sequences d'adn au moyen d'enzymes de restriction sensibles a l'hemi-methylation
US7459274B2 (en) 2004-03-02 2008-12-02 Orion Genomics Llc Differential enzymatic fragmentation by whole genome amplification
US8088581B2 (en) 2004-03-02 2012-01-03 Orion Genomics Llc Differential enzymatic fragmentation by whole genome amplification
US10822659B2 (en) 2006-03-31 2020-11-03 Affymetrix, Inc. Analysis of methylation using nucleic acid arrays
US9828640B2 (en) 2006-03-31 2017-11-28 Affymetrix, Inc. Analysis of methylation using nucleic acid arrays
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US11519036B2 (en) 2009-09-09 2022-12-06 The General Hospital Corporation Use of microvesicles in analyzing nucleic acid profiles
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US10793914B2 (en) 2010-08-31 2020-10-06 The General Hospital Corporation Cancer-related biological materials in microvesicles
WO2012031008A2 (fr) 2010-08-31 2012-03-08 The General Hospital Corporation Matières biologiques liées au cancer dans des microvésicules
US10988755B2 (en) 2010-11-10 2021-04-27 Exosome Diagnostics, Inc. Method for isolation of nucleic acid containing particles and extraction of nucleic acids therefrom
US12312582B2 (en) 2010-11-10 2025-05-27 Exosome Diagnostics, Inc. Method for isolation of nucleic acid containing particles and extraction of nucleic acids therefrom
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