EP2041316A2 - Procede de modification d'une macromolecule sans extraction prealable d'un echantillon - Google Patents

Procede de modification d'une macromolecule sans extraction prealable d'un echantillon

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Publication number
EP2041316A2
EP2041316A2 EP07810136A EP07810136A EP2041316A2 EP 2041316 A2 EP2041316 A2 EP 2041316A2 EP 07810136 A EP07810136 A EP 07810136A EP 07810136 A EP07810136 A EP 07810136A EP 2041316 A2 EP2041316 A2 EP 2041316A2
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EP
European Patent Office
Prior art keywords
dna
sample
macromolecule
bisulfite
modification
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Withdrawn
Application number
EP07810136A
Other languages
German (de)
English (en)
Other versions
EP2041316A4 (fr
Inventor
Christopher Steele
Abhijit Mazumder
Darin Oppenheimer
Sean Wuxiong Cao
Carrie Trust
George Green
Jyoti Mehrotra
Tatiana Vener
Shobha Varde
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.)
Janssen Diagnostics LLC
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Janssen Diagnostics LLC
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 Janssen Diagnostics LLC filed Critical Janssen Diagnostics LLC
Publication of EP2041316A2 publication Critical patent/EP2041316A2/fr
Publication of EP2041316A4 publication Critical patent/EP2041316A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • the present invention encompasses a method of modifying a macromolecule without prior extraction from a sample by converting the macromolecule in the sample with a chemical, removing or converting chemical intermediates, if necessary; and purifying the resulting modified macromolecule.
  • cytosines found in CpG islands located in promoter regions of various genes.
  • techniques were developed to discriminate methylated cytosines from unmethylated cytosines.
  • One method is to chemically treat DNA in such a way that the cytosines are converted to uracils while 5-methyl-cytosines are not significantly converted. Frommer et al. (1992). A systematic investigation on the critical parameters of the modification procedure has also been made. Grunau et al. (2001 ). The treated DNA may be used as template for methylation specific PCR (MSP).
  • MSP methylation specific PCR
  • DNA modification kits are commercially available, they convert purified genomic DNA with unmethylated cytosines into genomic lacking unmethylated cytosines but with additional uracils.
  • the treatment is a two-step chemical process consisting a deamination reaction facilitated by bisulfite and a desulfonation step facilitated by sodium hydroxide! Typically the deamination reaction is performed as a liquid and is terminated by incubation on ice followed by adding column binding buffer. Following solid phase binding and washing the DNA is eluted and the desulfonation reaction is performed in a liquid.
  • the present invention encompasses a method of modifying a macromolecule without prior extraction from a sample by converting the macromolecule in the sample with a chemical, removing or converting chemical intermediates, if necessary; and purifying the resulting modified macromolecule.
  • Figure 1 Shows that DNA modification in without isolation from a biological sample is equivalent to such modification after isolation.
  • the present invention encompasses a method of modifying a macromolecule without prior extraction from a sample by converting the macromolecule in the sample with a chemical, removing or converting chemical intermediates, if necessary; and purifying the resulting modified macromolecule.
  • the macromolecules can be any known in the art including, without limitation, DNA, RNA, cellular metabolites, lipids, carbohydrates and proteins.
  • the DNA can be any known in the art including, without limitation, viral, nucleic, mitochondrial, plastid, bacterial and synthetic.
  • the RNA can be any known in the art including, without limitation, rtRNA, tRNA, miRNA, rRNA and mRNA.
  • the cellular metabolite can be any known in the art including, without limitation, those produced by a metabolic cycle or enzymatic effects.
  • the lipid can be any known in the art including, without limitation, liposomes, cell membrane lipids, intracellular membrane lipids and extracellular lipids.
  • the carbohydrate can be any known in the art including, without limitation, protein-bound carbohydrates and nucleic acid-bound carbohydrates.
  • the protein can be any known in the art including, without limitation, intracellular and extracellular.
  • the modification is can be any known in the art including bisulfite and biotinylation of DNA or RNA, fluorination and methylation of RNA, heating, liposome formation, micelle formation, uni-layer formation and bilayer formation of lipids, oxidation, de-oxidation, amination and de-amination of carbohydrates and phosphorylation, dephosphorylation, methylation, biotinylation, amination, deamination, glycosylation and deglycosylation of proteins. 20070148670; Chuang et al. (2007); Emmerechts et al. (2007); Frommer et al. (1992); Grunau et al. (2001); Hurd et al. (2007); Jin et al. (2007); Oakeley (1999); Rathi et al. (2003); Rein et al. (1998); Sambrook et al. (2000); Wu et al. (2007).
  • the sample can be any known in the art including, without limitation, tissue, body fluid, a biopsy sample, and preserved tissue.
  • the tissue can be any known in the art including, without limitation, whole organs, dissected organs, epithelium, neural, gastrointestinal, muscle, cardiac, mucosal and endothelium.
  • the body fluid can be any known in the art including, without limitation, whole blood, plasma, urine, saliva, vitreous and serum.
  • the biopsy sample can be any known in the art including, without limitation, fine needle aspirate, tissue section and skin sample.
  • the preserved tissue can be any known in the art including, without limitation, fresh frozen, paraffin embedded and preserved in a preservation reagent.
  • the preservation reagent can be any known in the art including, without limitation, formalin, RNAlater®and dimethylsulfoxide.
  • the purification can be any known in the art including, without limitation, particle-based, precipitation, centrifugation, electrophoretic and charge switch.
  • the particle-based purification can be any known in the art including, without limitation, affinity, sizing and magnetic.
  • the sizing particle can be any known in the art including, without limitation, silica-based and diatomaceous earth.
  • the electrophoretic separation purification can be any known in the art including, without limitation, by size and/or charge.
  • the electrophoretic separation purification can be any known in the art including, without limitation, by a gel formed of low molecular weight polymers and/or capillary.
  • the present invention provides a rapid and efficient method for obtaining bisulfite modified DNA. The method described herein effectively eliminates numerous steps of the previous methods thus reducing possible error while producing superior results. In addition considerable time savings of four to five hours are also realized.
  • the present invention provides a method of extracting and modifying
  • DNA by obtaining a DNA sample; incubating the sample with an amount of a bisulfite and for a time and under conditions sufficient to convert at least ninety-five percent of the non-methylated cytosine residues in the DNA to uracil resides; binding the DNA in the sample to a column; washing the bound DNA to remove contaminants; incubating the column-bound DNA with a desulfonation reagent for a time and under conditions sufficient for desulfonation to occur; washing the bound DNA to remove the desulfonation reagent; and eluting the bisulfite modified DNA from the column.
  • the DNA can be at a concentration of from about 0.01 to about 30 ⁇ g and can be obtained by any method known in the art and can be purified DNA or DNA obtained directly from a cell lysate.
  • the cell lysate can be formed from any suitable tissue by any method known in the art and directly treated with a bisulfite reagent.
  • Cell lysis can be by for instance, proteinase and/or high salt concentration and/or detergent, sonication, freeze-thaw treatment or mechanical disruption. Any cell sample is suitable for use herein and can be obtained from tissue, body fluid, biopsy sample or preserved tissue.
  • the bisulfite reagent can be any known in the art, including, without limitation, sodium bisulfite or meta bisulfite. Other reagents are discussed for instance in US patent publications 20050089898, 20050095623 and 20050153308.
  • the incubation conditions of step b are about 1-16 hours at 50-95 0 C with or without Thermocy cling.
  • Thermocycling can be for instance 3 hours at 70 0 C, 1 hour at 90 ⁇ C or cycling between 50 0 C and 95°C.
  • the column can be any known in the art, preferably, it is silica-based or diatomaceous earth.
  • the desulfonation can be by any method known in the art and is preferably performed with sodium hydroxide and an alcohol.
  • the alcohol is isopropanol or ethanol. More preferably, when the column is silica-based, the alcohol is ethanol and when the column is diatomaceous earth, the alcohol is isopropanol.
  • the desulfonation preferably occurs from about 0-30, preferably about 5-15 and more preferably about 15 minutes at about 0 0 C to about 50 0 C.
  • the temperature is about room temperature.
  • the modified macromolecule can be eluted by any method known in the art, including, without limitation with water or a suitable buffer.
  • FFPE FFPE
  • the efficiency of the procedure is assayed using quantitative PCR and ⁇ -Actin using GSTP1 as markers.
  • the ⁇ -Actin promoter is not methylated and the marker is designed to serve as a control for the modification procedure.
  • the Ct value produced by this marker is reflective of the number of genome equivalents added to the assay.
  • the GSTP1 promoter is methylated epigenetically and may be reflective of a cancerous state. Therefore the GHSTP1 marker Ct value is more variable and usually greater than the ⁇ -Actin Ct value.
  • the Prostate FFPE blocks were obtained from Asterand.
  • the "Zymo" treatment refers commercially available DNA modification kit sold as EZ DNA Methylatio ⁇ Kit from Zymo Research.
  • a 2 Step procedure refers to two separate procedures that use a DNA purification kit (Qiagen QiaAmp mini DBNA purification kit) and a DNA modification kit such as the Zymo kit.
  • the Zymo 1 step procedure is identical to the ID procedure until the 3M NaOH step where the Zymo DNA modification procedure is followed replacing the 3M NaOH with M-Dilution buffer.
  • results shown in Table 1 using prostate FFPE blocks indicate the opposite results from the cell cultures with the Zymo 2 Step producing lower Ct values then the 1 step method.
  • the ⁇ -Actin marker results indicate that the methods are significantly different with a P value of 0.021 while the GSTP1 results suggest that they maybe different with a P value of 0.054.
  • the different results suggested that the extraction buffer for cell culture might not be sufficient to lysis tissue blocks.
  • Table 4 results compares the ID 1 Step and the Zymo 1 Step methods using a more aggressive extraction buffer switching the Tween to SDS while using prostate tissue blocks.
  • the ID 1 Step method once again shows superior results suggesting that the failure to produce superior results in Table 3 was due to the extraction buffer.
  • the ⁇ - Actin marker results indicates that the methods are significantly different with a P value of 0.0008 while the GSTP1 results suggest that they maybe different with a P value of 0.056. Since the QPCR assay method only has 40 cycles, once an assay fails to produce Ct's then it is not significant to compare with assays that produce Ct's. If the two samples that had assays that failed then the P values would be 0.0025, which indicates the methods using the GSTP1 marker are significantly different.
  • Table 4 Results comparing the 1 Step procedure to the Zymo 1 Step rocedure usin Prostate FFPE blocks.
  • the purpose of this experiment was to compare the DEM kit versus the Zymo EZ Modification kit on purified DNA obtained from LnCAP cells and urine. Ten normalized random samples were divided between the two kits
  • ANOVA analysis indicates a P value of 0.663 and a Paired T-Test indicates that there is no statistical difference between DEM and Zymo for GSTP1.
  • the binding of crude lysate containing DNA or purified genomic DNA is uniquely bound to the silica-gel based column utilizing the high concentration of salt that is present from the bisulfite conversion.
  • Ethanol is added to the sample prior to binding only to dissolve the conversion reagent.
  • microfuge tube containing (up to) 5 10 micron FFPE block slices of a formalin fixed paraffin embedded cell culture pellet which is known to express GSTP1 hyper Methylation.
  • the FFPE slices require deparaffination:
  • DNA (ATCC) in a total volume of 225 ⁇ l TE, plus 27.5 ⁇ l of a 3.0 M NaOH solution. Incubate 10 minutes 37°C.
  • Example 4 A modified protocol for fast and efficient bisulfite modification of genomic DNA
  • the EZ DNA Methylation Kit is provided by Zymo Research (Orange, CA) to perform bisulfite modification of DNA. As per manufacturer's recommendation the DNA sample to be modified is incubated with the bisulfite conversion reagent at 5O 0 C for 12 — 16 hrs. These conditions have been modified to generate comparable quality bisulfite converted DNA in much less time. Several temperatures for different times were tested and demonstrated that incubation of DNA sample with bisulfite conversion reagent at 70 c C for 1 - 3 hr provides efficient bisulfite modification comparable to modification conditions recommended in the kit. The data below show methylation specific PCR analysis with DNA samples incubated with bisulfite reagent at different temperatures for different times.
  • Figure 1 shows that Veridex modified protocol of conversion at 7O 0 C, 2 or 3hr is equivalent to manufacturer recommended 50 0 C for 16 hrs. This innovation makes this procedure much faster.
  • Extraction of genomic DNA and its bisulfite modification prior to being used in a MSP reaction comprise very significant upstream procedures that are part of this in vitro diagnostic assay. These procedures can be time consuming involving many tedious steps and could also increase chances of sample contamination.
  • a lysis buffer 10 mM Tris pH 8.0, 150 mM NaCI, 2 mM EDTA, 0.5% SDS including proteinase K and a bisulfite modification kit
  • FFPE tissue (Biopsies) (5 x 10 ⁇ sections) are placed in Eppendorf tubes.
  • TNES lysis buffer (10 mM Tris pH 8.0, 150 mM NaCI, 2 mM EDTA 1 0.5% SDS) and suspend the tissue by flicking the tube.
  • CT Conversion Reagent (after briefly spinning) to each sample and vortex lightly (The sample may turn cloudy). Spin the sample briefly. Incubate the sample at 70 0 C for 3 hr with the heating block ⁇ shaking at 1100 rpm) covered with aluminum foil. (The CT Conversion Reagent is light sensitive, so try to minimize reaction's exposure to light).
  • the protocol described above excludes the use of a DNA purification kit prior to bisulfite modification, thereby, reducing sample processing times, preventing DNA losses during purification, reducing cost, and reducing chances of contamination.
  • Table 6 shows that using the tissue lysate directly for bisulfite modification of DNA gives comparable and even lower Cts than purified DNA using Qiagen DNA isolation kit. Thus further purification of DNA is not required prior to DNA modification using ZymoResearch EZ DNA methylation kit. Also, the data show that combining TNES/PK digestion and EZ DNA methylation kit yields more DNA sample. Table 6
  • Table 7 shows that direct lysate from FFPE biopsy tissue can be used successfully for downstream DNA modification with comparable and even better results as compared to using Qiagen DNA isolation kit, thus avoiding unnecessary DNA purification steps and losses.
  • DNA methylation assay is developed to be used on patient samples such as archived formalin-fixed, paraffin-embedded tissues, freshly collected urine and blood samples that comprise an invaluable resource for translational studies of cancer and a variety of other diseases.
  • Sample processing is a key upstream part of this diagnostic assay.
  • DNA is purified from these sample types by using standard phenol-chloroform extraction or column based procedures and then subjected to bisulfite modification procedures.
  • TNES protocol quickly and efficiently extracts and bisulfite modifies genomic DNA by using the deproteinized lysed tissue extract or lysed cells from urine sediment and directly using this with a commercially, available DNA methylation kit such as ZymoResearch EZ for downstream bisulfite modification without any further purification steps.
  • ZymoResearch EZ DNA methylation kit
  • the . present invention improves multiplex PCR assay performance by minimizing loss during additional purification steps.
  • Urine is placed into a labeled 50 ml Falcon polypropylene tube and LNCap cells (10,000 cells/50 ml) representing shedded tumor cells in a prostate cancer patient are spiked per tube. Then the cells and particulates are pelleted by centrifugation at 3,000 x g at 4°C.
  • the pellet is resuspended in a smaller volume of 1 ml of cold PBS by gently pipetting up and down with a Pipetman. Once the pellet is suspended, then transfer from the 50 ml tube to a 1.5 ml microcentrifuge tube. With an additional 0.4 ml PBS, rinse the tip and the 50 ml tube to recover as much of pellet as possible and combine with the original 1 ml in the 1.5 ml tube.
  • TNES lysis buffer (10 mM Tris pH 8.0, 150 mM NaCI, 2 mM EDTA, 0.5% SDS), incubated ⁇ g 56°C for 30 min. and then stored at -2O 0 C until processing with bisulfite modification kit (see below, part II). In case of cells only, 20 ⁇ l TNES buffer was added to 20 ⁇ l cell suspension.
  • the EZ DNA Methylation Kit is provided by Zymo Research .(Orange, CA) to perform bisulfite modification of DNA. As per manufacturer's recommendation the DNA sample to be modified is incubated with the bisulfite conversion reagent at 50 0 C for 12 - 16 hrs. These conditions have now been modified to generate comparable quality bisulfite converted DNA in much less time. Several temperatures and different times were tested and it was demonstrated that incubation of DNA sample with bisulfite conversion reagent at 70 0 C for 1 — 3 hr provides efficient bisulfite modification comparable to modification conditions recommended in the kit
  • the protocol is as follows for processing urine samples:
  • M-Wash Buffer (Prepare before starting using the kit) Preparation of M-Wash buffer: Add 24 ml absolute ethanol to the M-Wash buffer Concentrate to make the final M-Wash buffer for D5001 (Use 96 ml Ethanol for D5002).
  • DNA modification procedure a. Add 5 ⁇ l of M-Dilution Buffer directly to 45 ⁇ l of urine lysate or for the higher sample volume scale up M-Dilution Buffer and urine crude lysate proportionally. For instance, for 150 ⁇ l urine lysate add 20 ⁇ l M-dilution buffer and 30 ⁇ l water (total of 200 ⁇ l).
  • b. Mix sample by flicking or pipetting up and down. Spin the sample briefly.
  • this step is repeated by adding supernatant from each aliquoted tube one at a time on the column and centrifuging until all of the sample is loaded onto the column.
  • d Add 200 ⁇ l of M-Wash Buffer to the column.
  • e Centrifuge at maximum speed for 15 - 30 seconds. Discard the flow-through.
  • the protocol described above excludes the use of a DNA purification kit prior to bisulfite modification, thereby, reducing sample processing times, preventing DNA losses during purification, reducing cost, and reducing chances of contamination).
  • Table 8 shows end results from MSP assay on Cepheid Smart Cycler with DNA samples processed by TNES protocol from 50 ml of urine. Better ⁇ -actin and GSTP1 Cts (up to 3 Cts lower) are observed using above described TNES/PK digestion protocol over use of purified DNA using commercially available Qiagen DNA isolation kit (QiAmp Viral RNA kit). Thus further purification of DNA is not required prior to DNA modification using this method. Also, the data show that combining TNES/PK digestion and EZ DNA methylation kit yields more DNA sample.
  • TNES extraction protocol results are even more compelling on serial dilutions of LNCaP prostate cells (range of 10,000 to 100 spiked per 50 ml pooled urine from healthy donors). Combined protocol for DNA extraction followed directly by bisulfite modification allows to improve sensitivity of the prostate methylation assay by 10 fold (Table 9) as compared with two commercial kits combined. TNES protocol allows detection of 100 cells per 50 mi urine, the level which is undetectable by Qiagen protocol using both Ct value and copy number analysis.

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Abstract

La présente invention concerne un procédé de modification d'une macromolécule sans extraction préalable d'un échantillon par la conversion de la macromolécule dans l'échantillon avec un produit chimique, l'élimination ou la conversion d'intermédiaires chimiques, le cas échéant, et la purification de la macromolécule modifiée ainsi obtenue.
EP07810136A 2006-06-29 2007-06-29 Procédé de modification d'une macromolécule sans extraction préalable d'un échantillon Withdrawn EP2041316A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US81787606P 2006-06-29 2006-06-29
US82879006P 2006-10-10 2006-10-10
PCT/US2007/015329 WO2008002680A2 (fr) 2006-06-29 2007-06-29 Procédé de modification d'une macromolécule sans extraction préalable d'un échantillon

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EP2041316A2 true EP2041316A2 (fr) 2009-04-01
EP2041316A4 EP2041316A4 (fr) 2009-12-09

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EP (1) EP2041316A4 (fr)
JP (1) JP2009542216A (fr)
KR (1) KR20090036121A (fr)
BR (1) BRPI0713927A2 (fr)
CA (1) CA2656327A1 (fr)
IL (1) IL196198A0 (fr)
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US8298852B2 (en) 2008-12-29 2012-10-30 Jusung Engineering Co., Ltd. Thin film type solar cell and method for manufacturing the same
EP2407540A1 (fr) * 2010-07-15 2012-01-18 Qiagen GmbH Procédé de purification d'un acide nucléique cible
WO2014120918A1 (fr) * 2013-01-30 2014-08-07 Exact Sciences Corporation Traitement de vaisseau échantillonnés
CN107821989B (zh) * 2017-12-04 2020-11-17 南京农业大学 一种提高类pse鸡胸肉肌原纤维蛋白凝胶品质的糖基化方法
WO2022066844A1 (fr) * 2020-09-24 2022-03-31 Case Western Reserve University Compositions et procédés de préservation de la méthylation de l'adn

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067775A2 (fr) * 2003-01-30 2004-08-12 Epigenomics Ag Technique de detection de configurations de methylation de cytosine a haute sensibilite

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004067775A2 (fr) * 2003-01-30 2004-08-12 Epigenomics Ag Technique de detection de configurations de methylation de cytosine a haute sensibilite

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRUNAU C; CLARK S J; ROSENTHAL A: "Bisulfite genomic sequencing: systematic investigation of critical experimental parameters" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 29, no. 13, 1 January 2001 (2001-01-01), pages E65-1, XP002241346 ISSN: 0305-1048 *
OLEK A; ET AL: "A modified an improved method for bisulphite based cytosine methylation analysis" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 24, no. 24, 1 January 1996 (1996-01-01), pages 5064-5066, XP002106408 ISSN: 0305-1048 *
See also references of WO2008002680A2 *
SHIRAISHI MASAHIKO; HAYATSU HIKOYA: "High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation." DNA RESEARCH : AN INTERNATIONAL JOURNAL FOR RAPID PUBLICATION OF REPORTS ON GENES AND GENOMES 31 DEC 2004, vol. 11, no. 6, 31 December 2004 (2004-12-31), pages 409-415, XP002545192 ISSN: 1340-2838 *

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MX2009000122A (es) 2009-01-26
WO2008002680A3 (fr) 2008-11-27
BRPI0713927A2 (pt) 2013-01-08
IL196198A0 (en) 2009-09-22
WO2008002680A2 (fr) 2008-01-03
KR20090036121A (ko) 2009-04-13
CA2656327A1 (fr) 2008-01-03
EP2041316A4 (fr) 2009-12-09
JP2009542216A (ja) 2009-12-03

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