EP1292707A2 - Oligonucleotides ou oligomeres de pna, et procede de detection parallele de l'etat de methylation d'un adn genomique - Google Patents
Oligonucleotides ou oligomeres de pna, et procede de detection parallele de l'etat de methylation d'un adn genomiqueInfo
- Publication number
- EP1292707A2 EP1292707A2 EP01927582A EP01927582A EP1292707A2 EP 1292707 A2 EP1292707 A2 EP 1292707A2 EP 01927582 A EP01927582 A EP 01927582A EP 01927582 A EP01927582 A EP 01927582A EP 1292707 A2 EP1292707 A2 EP 1292707A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- artificial sequence
- dna
- oligonucleotide
- description
- cgtacg
- 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.)
- Withdrawn
Links
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 421
- 238000000034 method Methods 0.000 title claims abstract description 76
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 230000011987 methylation Effects 0.000 title claims abstract description 30
- 238000007069 methylation reaction Methods 0.000 title claims abstract description 30
- 239000012634 fragment Substances 0.000 claims abstract description 28
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims abstract description 21
- 108020004414 DNA Proteins 0.000 claims description 454
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 74
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 50
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 32
- 229940104302 cytosine Drugs 0.000 claims description 31
- 238000009396 hybridization Methods 0.000 claims description 31
- 239000000523 sample Substances 0.000 claims description 31
- 229940113082 thymine Drugs 0.000 claims description 25
- 229930024421 Adenine Natural products 0.000 claims description 24
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 24
- 229960000643 adenine Drugs 0.000 claims description 24
- 230000003321 amplification Effects 0.000 claims description 24
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 24
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 16
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 16
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 13
- 238000003752 polymerase chain reaction Methods 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 229940035893 uracil Drugs 0.000 claims description 7
- 239000011159 matrix material Substances 0.000 claims description 6
- 238000000018 DNA microarray Methods 0.000 claims description 5
- WBZKQQHYRPRKNJ-UHFFFAOYSA-L disulfite Chemical compound [O-]S(=O)S([O-])(=O)=O WBZKQQHYRPRKNJ-UHFFFAOYSA-L 0.000 claims description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 238000002966 oligonucleotide array Methods 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 3
- 230000030933 DNA methylation on cytosine Effects 0.000 claims description 3
- 238000003795 desorption Methods 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 claims description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- 108010043461 Deep Vent DNA polymerase Proteins 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 241000722234 Pseudococcus Species 0.000 claims description 2
- 241000205156 Pyrococcus furiosus Species 0.000 claims description 2
- 241000205192 Pyrococcus woesei Species 0.000 claims description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 2
- 229910000831 Steel Inorganic materials 0.000 claims description 2
- 108010006785 Taq Polymerase Proteins 0.000 claims description 2
- 101000865057 Thermococcus litoralis DNA polymerase Proteins 0.000 claims description 2
- 229910052782 aluminium Inorganic materials 0.000 claims description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 claims description 2
- 239000011324 bead Substances 0.000 claims description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims description 2
- 230000033228 biological regulation Effects 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 229910052742 iron Inorganic materials 0.000 claims description 2
- 229910052759 nickel Inorganic materials 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 229910052710 silicon Inorganic materials 0.000 claims description 2
- 239000010703 silicon Substances 0.000 claims description 2
- 229910052709 silver Inorganic materials 0.000 claims description 2
- 239000004332 silver Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000010959 steel Substances 0.000 claims description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 2
- 229940104230 thymidine Drugs 0.000 claims description 2
- 235000012431 wafers Nutrition 0.000 claims description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 claims 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 claims 1
- 238000011156 evaluation Methods 0.000 claims 1
- 239000000758 substrate Substances 0.000 claims 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 9
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 108050007957 Cadherin Proteins 0.000 description 3
- 108010054218 Factor VIII Proteins 0.000 description 3
- 101000933342 Homo sapiens BMP/retinoic acid-inducible neural-specific protein 1 Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000001973 epigenetic effect Effects 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 229940079826 hydrogen sulfite Drugs 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000002516 radical scavenger Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012491 analyte Substances 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- HWPZZUQOWRWFDB-UHFFFAOYSA-N 1-methylcytosine Chemical compound CN1C=CC(N)=NC1=O HWPZZUQOWRWFDB-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000001698 laser desorption ionisation Methods 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000037425 regulation of transcription Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
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- 238000012552 review Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
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- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
Definitions
- the second variant is based on partial chemical cleavage of total DNA, modeled on a Maxam-Gilbert sequencing reaction, ligation of adapters to the ends thus generated, amplification with generic primers and separation on a gel electrophoresis.
- This method defined areas up to the size of less than a thousand base pairs can be examined.
- the process is so complicated and unreliable that it is practically no longer used (Ward, C. et al., J. Biol. Chem. 265, 3030-3033).
- the present invention describes a method and a set of oligonucleotides or PNA oligomers for the parallel detection of the methylation state : ⁇ ( ⁇ ) 90ivi99i ⁇ ( ⁇ ): ⁇ ( ⁇ ) 90ivi990 ⁇ ( ⁇ ) * ⁇ ( ⁇ ) 9 ⁇ vi99i ⁇ ( ⁇ )
- a genomic DNA sample is chemically treated in such a way that at the 5'-position unmethylated cytosine bases are converted into uracil, thymine or another base which is unlike cytosine in terms of hybridization behavior.
- the treatment of genomic DNA with bisulfite (bisulfite, disulfite) and subsequent alkaline hydrolysis, which leads to a conversion of unmethylated cytosine nucleobases into uracil, is preferably used for this purpose.
- more than 26 different oligonucleotides are used simultaneously to produce a complex amplificate.
- the products obtained after the amplification are separated by gel electrophoresis and the fragments which are smaller than 2000 base pairs or smaller than any limit below
- the CpG dinucleotides contained in the amplified fragments are also examined completely or partially by hybridization of the fragments already provided with a detectable label in the amplification to a set of PNA oligomers which comprises at least two of the following sequences:
- W one of the bases adenine (A) or thymine (T)
- the first step is a genomic sequence using bisulfite
- Figure 1 Here a high-density DNA chip is shown after hybridization.
- the false color image is shown as it is generated after scanning. Contrary to the black and white illustration shown here, the scanner turns a colored one
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
L'invention concerne un procédé et un jeu d'oligonucléotides ou d'oligomères de PNA pour la détection parallèle de l'état de méthylation d'un ADN génomique. Selon l'invention, l'ADN est d'abord traité avec du bisulfites et cet ADN chimiquement modifié est ensuite fragmenté. Dans l'étape suivante, on procède à l'amplification de différents fragments avec des amorces synthétiques. Les produits de l'amplification sont hybridés avec des oligonucléotides d'une séquence connue et enfin détectés.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10013847 | 2000-03-15 | ||
| DE10013847A DE10013847A1 (de) | 2000-03-15 | 2000-03-15 | Oligonukleotide oder PNA-Oligomere und Verfahren zur parallelen Detektion des Methylierungszustandes genomischer DNA |
| PCT/DE2001/001089 WO2001068910A2 (fr) | 2000-03-15 | 2001-03-15 | Oligonucleotides ou oligomeres de pna, et procede de detection parallele de l'etat de methylation d'un adn genomique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1292707A2 true EP1292707A2 (fr) | 2003-03-19 |
Family
ID=7635678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP01927582A Withdrawn EP1292707A2 (fr) | 2000-03-15 | 2001-03-15 | Oligonucleotides ou oligomeres de pna, et procede de detection parallele de l'etat de methylation d'un adn genomique |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US20050202420A1 (fr) |
| EP (1) | EP1292707A2 (fr) |
| AU (1) | AU2001254601A1 (fr) |
| DE (1) | DE10013847A1 (fr) |
| WO (1) | WO2001068910A2 (fr) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003144172A (ja) * | 2001-11-16 | 2003-05-20 | Nisshinbo Ind Inc | メチル化検出用オリゴヌクレオチド固定化基板 |
| EP1693468A1 (fr) | 2005-02-16 | 2006-08-23 | Epigenomics AG | Procédé de détection de l'état de méthylation d'un acide polynucléique |
| US7932027B2 (en) | 2005-02-16 | 2011-04-26 | Epigenomics Ag | Method for determining the methylation pattern of a polynucleic acid |
| WO2006113770A1 (fr) | 2005-04-15 | 2006-10-26 | Epigenomics Ag | Procede destine a fournir des fragments d'adn derives d'un echantillon a distance |
| US7820385B2 (en) | 2006-03-22 | 2010-10-26 | The United States Of America As Represented By The Department Of Health And Human Services, Centers For Disease Control And Prevention | Method for retaining methylation pattern in globally amplified DNA |
| US8084734B2 (en) * | 2006-05-26 | 2011-12-27 | The George Washington University | Laser desorption ionization and peptide sequencing on laser induced silicon microcolumn arrays |
| US8110796B2 (en) | 2009-01-17 | 2012-02-07 | The George Washington University | Nanophotonic production, modulation and switching of ions by silicon microcolumn arrays |
| US9490113B2 (en) * | 2009-04-07 | 2016-11-08 | The George Washington University | Tailored nanopost arrays (NAPA) for laser desorption ionization in mass spectrometry |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04500609A (ja) * | 1989-06-29 | 1992-02-06 | ギスト ブロカデス ナームローゼ フェンノートチャップ | 工業的応用条件下で安定性が減少した酵素変異体 |
| US6017704A (en) * | 1996-06-03 | 2000-01-25 | The Johns Hopkins University School Of Medicine | Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids |
| DE19754482A1 (de) * | 1997-11-27 | 1999-07-01 | Epigenomics Gmbh | Verfahren zur Herstellung komplexer DNA-Methylierungs-Fingerabdrücke |
| AU3671099A (en) * | 1998-04-29 | 1999-11-16 | Trustees Of Boston University | Methods and compositions pertaining to pd-loops |
| AU8229598A (en) * | 1998-07-02 | 2000-01-24 | Imperial Cancer Research Technology Limited | Tumour suppressor gene dbccr1 at 9q32-33 |
| US6107091A (en) * | 1998-12-03 | 2000-08-22 | Isis Pharmaceuticals Inc. | Antisense inhibition of G-alpha-16 expression |
| US6136603A (en) * | 1999-03-26 | 2000-10-24 | Isis Pharmaceuticals Inc. | Antisense modulation of interleukin-5 signal transduction |
-
2000
- 2000-03-15 DE DE10013847A patent/DE10013847A1/de not_active Ceased
-
2001
- 2001-03-15 WO PCT/DE2001/001089 patent/WO2001068910A2/fr not_active Ceased
- 2001-03-15 US US10/221,878 patent/US20050202420A1/en not_active Abandoned
- 2001-03-15 AU AU2001254601A patent/AU2001254601A1/en not_active Abandoned
- 2001-03-15 EP EP01927582A patent/EP1292707A2/fr not_active Withdrawn
Non-Patent Citations (2)
| Title |
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| None * |
| See also references of WO0168910A3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| DE10013847A1 (de) | 2001-09-27 |
| US20050202420A1 (en) | 2005-09-15 |
| WO2001068910A3 (fr) | 2003-01-03 |
| WO2001068910A2 (fr) | 2001-09-20 |
| AU2001254601A1 (en) | 2001-09-24 |
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