Methylated DNA Immunoprecipitation and High-Throughput Sequencing (Medip-Seq) Using Low Amounts of Genomic DNA
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Epigenome Chaos: Stochastic and Deterministic DNA Methylation Events Drive Cancer Evolution
cancers Review Epigenome Chaos: Stochastic and Deterministic DNA Methylation Events Drive Cancer Evolution Giusi Russo 1, Alfonso Tramontano 2, Ilaria Iodice 1, Lorenzo Chiariotti 1 and Antonio Pezone 1,* 1 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli “Federico II”, 80131 Naples, Italy; [email protected] (G.R.); [email protected] (I.I.); [email protected] (L.C.) 2 Department of Precision Medicine, University of Campania “L. Vanvitelli”, 80138 Naples, Italy; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +39-081-746-3614 Simple Summary: Cancer is a group of diseases characterized by abnormal cell growth with a high potential to invade other tissues. Genetic abnormalities and epigenetic alterations found in tumors can be due to high levels of DNA damage and repair. These can be transmitted to daughter cells, which assuming other alterations as well, will generate heterogeneous and complex populations. Deciphering this complexity represents a central point for understanding the molecular mechanisms of cancer and its therapy. Here, we summarize the genomic and epigenomic events that occur in cancer and discuss novel approaches to analyze the epigenetic complexity of cancer cell populations. Abstract: Cancer evolution is associated with genomic instability and epigenetic alterations, which contribute to the inter and intra tumor heterogeneity, making genetic markers not accurate to monitor tumor evolution. Epigenetic changes, aberrant DNA methylation and modifications of chromatin proteins, determine the “epigenome chaos”, which means that the changes of epigenetic traits are Citation: Russo, G.; Tramontano, A.; randomly generated, but strongly selected by deterministic events. -
The International Human Epigenome Consortium (IHEC): a Blueprint for Scientific Collaboration and Discovery
The International Human Epigenome Consortium (IHEC): A Blueprint for Scientific Collaboration and Discovery Hendrik G. Stunnenberg1#, Martin Hirst2,3,# 1Department of Molecular Biology, Faculties of Science and Medicine, Radboud University, Nijmegen, The Netherlands 2Department of Microbiology and Immunology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada V6T 1Z4. 3Canada’s Michael Smith Genome Science Center, BC Cancer Agency, Vancouver, BC, Canada V5Z 4S6 #Corresponding authors [email protected] [email protected] Abstract The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalogue of high-resolution reference epigenomes of major primary human cell types. The studies now presented (cell.com/XXXXXXX) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic data sets to gain insights in the epigenetic control of cell states relevant for human health and disease. One of the great mysteries in developmental biology is how the same genome can be read by cellular machinery to generate the plethora of different cell types required for eukaryotic life. As appreciation grew for the central roles of transcriptional and epigenetic mechanisms in specification of cellular fates and functions, researchers around the world encouraged scientific funding agencies to develop an organized and standardized effort to exploit epigenomic assays to shed additional light on this process (Beck, Olek et al. 1999, Jones and Martienssen 2005, American Association for Cancer Research Human Epigenome Task and European Union 2008). In March 2009, leading scientists and international health research funding agency representatives were invited to a meeting in Bethesda (MD, USA) to gauge the level of interest in an international epigenomics project and to identify potential areas of focus. -
Three Optimized Workflows for Cpg Island Methylation Profiling
APPLICATION NOTE Three Optimized Workflows for CpG Island Methylation Profiling Three Optimized Workflows for CpG Island Methylation Profiling DNA methylation is involved in the Discovery regulation of many cellular processes SOLiD™ Do you know your candidate gene? including X chromosome inactivation, No System chromosome stability, chromatin structure, Yes embryonic development, and transcription. Validation/Screening with Capillary Electrophoresis Do you know your candidate region or In mammals only the 5' carbons of the methylation state of that region? cytosines adjacent to guanines (CpG Yes No dinucleotides: cytosine-phospho-guanine) MSMSA Bisulfite Conversion Bisulfite Conversion are substrates for DNA methylation. (MethylSEQr™ Kit) (MethylSEQr™ Kit) Importance of CpG Islands Are there a high number Bisulfite Specific (BSP) CpG islands are 300−3000 base pair of poly T or A, or large Primer Design fragment size (>300 bps)? (Methyl Primer® Express) stretches of DNA that are CG rich, and are often found within unmethylated Yes No regions of promoters. Profiling of CpG Clone based Sequencing Direct PCR Sequencing island methylation indicates that some Fluorescent BSP PCR genes are more frequently methylated Bisulfite Specific (BSP) Primer Design Bisulfite Specific (BSP) Primer Design Data Analysis (Methyl Primer® Express) (Methyl Primer® Express) than others [1]. Hence, CpG islands play a (GeneMapper® Software v. 4.0) critical role in regulating gene expression. CpG islands found outside promoter BSP PCR BSP PCR Identification of a candidate region or the No regions are commonly methylated, and methylation state of Yes that region? are believed to be responsible for silencing Cloning Yes transcription of repetitive sequences Cycle Sequencing Direct PCR Cycle Sequencing Are there a high number and parasitic sequence elements, such of poly T or A, or large No as viral DNA. -
Epigenome-Wide Association Study (EWAS) on Lipids: the Rotterdam Study Kim V
Braun et al. Clinical Epigenetics (2017) 9:15 DOI 10.1186/s13148-016-0304-4 RESEARCH Open Access Epigenome-wide association study (EWAS) on lipids: the Rotterdam Study Kim V. E. Braun1, Klodian Dhana1, Paul S. de Vries1,2, Trudy Voortman1, Joyce B. J. van Meurs3,4, Andre G. Uitterlinden1,3,4, BIOS consortium, Albert Hofman1,5, Frank B. Hu5,6, Oscar H. Franco1 and Abbas Dehghan1* Abstract Background: DNA methylation is a key epigenetic mechanism that is suggested to be associated with blood lipid levels. We aimed to identify CpG sites at which DNA methylation levels are associated with blood levels of triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and total cholesterol in 725 participants of the Rotterdam Study, a population-based cohort study. Subsequently, we sought replication in a non-overlapping set of 760 participants. Results: Genome-wide methylation levels were measured in whole blood using the Illumina Methylation 450 array. Associations between lipid levels and DNA methylation beta values were examined using linear mixed-effect models. All models were adjusted for sex, age, smoking, white blood cell proportions, array number, and position on array. A Bonferroni-corrected p value lower than 1.08 × 10−7 was considered statistically significant. Five CpG sites annotated to genes including DHCR24, CPT1A, ABCG1,andSREBF1 were identified and replicated. Four CpG sites were associated with triglycerides, including CpG sites annotated to CPT1A (cg00574958 and cg17058475), ABCG1 (cg06500161), and SREBF1 (cg11024682). Two CpG sites were associated with HDL-C, including ABCG1 (cg06500161) and DHCR24 (cg17901584). No significant associations were observed with LDL-C or total cholesterol. -
DNA Methylation Analysis: Choosing the Right Method
biology Review DNA Methylation Analysis: Choosing the Right Method Sergey Kurdyukov 1,* and Martyn Bullock 2 Received: 8 July 2015; Accepted: 22 December 2015; Published: 6 January 2016 Academic Editor: Melanie Ehrlich 1 Genomics Core facility, Kolling Institute of Medical Research, University of Sydney, Sydney 2065, Australia 2 Cancer Genetics Laboratory, Kolling Institute of Medical Research, University of Sydney, Sydney 2065, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-299-264-756 Abstract: In the burgeoning field of epigenetics, there are several methods available to determine the methylation status of DNA samples. However, choosing the method that is best suited to answering a particular biological question still proves to be a difficult task. This review aims to provide biologists, particularly those new to the field of epigenetics, with a simple algorithm to help guide them in the selection of the most appropriate assay to meet their research needs. First of all, we have separated all methods into two categories: those that are used for: (1) the discovery of unknown epigenetic changes; and (2) the assessment of DNA methylation within particular regulatory regions/genes of interest. The techniques are then scrutinized and ranked according to their robustness, high throughput capabilities and cost. This review includes the majority of methods available to date, but with a particular focus on commercially available kits or other simple and straightforward solutions that have proven to be useful. Keywords: DNA methylation; 5-methylcytosine; CpG islands; epigenetics; next generation sequencing 1. Introduction DNA methylation in vertebrates is characterized by the addition of a methyl or hydroxymethyl group to the C5 position of cytosine, which occurs mainly in the context of CG dinucleotides. -
High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early-Onset and Familial Risk Breast Cancer Patients
Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early- Onset and Familial Risk Breast Cancer Patients Justin Chen1*, Maria K. Haanpää2*, Joshua J. Gruber1,2, Natalie Jäger1, James M. Ford1,2ǂ, and Michael P. Snyder1ǂ 1Department of Genetics 2Department of Medicine, Oncology Division Stanford University, Stanford, CA 94305, USA. * These authors contributed equally to this work. ǂ Co-corresponding authors Running Title: DNA Methylation and High-Risk Breast Cancer Keywords: DNA Methylation; Breast Cancer; Epigenetics; Cancer Predisposition; Allelic Methylation Additional Information: Financial Support: This work used the Genome Sequencing Service Center by Stanford Center for Genomics and Personalized Medicine Sequencing Center, supported by the grant award NIH S10OD020141. MKH is supported by grants from Sigrid Juselius Foundation, Orion Research Foundation, Päivikki ja Sakari Sohlberg Foundation and Instrumentarium Science Foundation. JJG was supported by fellowships from the Jane Coffin Childs Memorial Fund for Medical Research, Stanford Cancer Institute and Susan G. Komen Foundation, as well as funding from ASCO, the Conquer Cancer Foundation and the Breast Cancer Research Foundation. NJ was supported by an EMBO Long-Term Fellowship (ALTF 325-2014). JMF is supported by the BRCA Foundation and the Breast Cancer Research Foundation. MPS is supported by grants from the NIH including a Centers of Excellence in Genomic Science award (5P50HG00773504). Correspondence: Michael P. Snyder James M. Ford Department of Genetics 269 Campus Dr. Stanford University School of Medicine CCSR Room 1115 300 Pasteur Dr. -
Sulfur Dioxide and Some Sulfites, Bisulfites and Metabisulfites
SULFUR DIOXIDE AND SOME SULFITES, BISULFITES AND METABISULFITES 1. Exposure Data 1.1 Chemical and physical data 1.1.1 Synonyms and structural and molecular data Sulfr dioxi Chem. Abstr. Serv Reg. No.: 7446-09-5 Replaced CAS Nos.: 8014-94-6; 12396-99-5; 83008-56-4; 89125-89-3 Chem. Abstr. Name; Sulfur dioxide IUPAC Systematic Name: Sulfur dioxide Synonyms: Sulfurous acid anhydride; sulfurous anhydride; sulfurous oxide; sulfur oxide (S02); sulfur superoxide; sulphur dioxide 0=8=0 S02 MoL. wt: 64.07 Sodium sulfte Chem. Abstr. Serv Reg. No.: 7757-83-7 Altemate CAS No.: 10579-83-6 Replaced CAS No.: 68135-69-3 Chem. Abstr. Name: Sulfurous acid, di sodium salt IUPAC Systematic Name: Sulfurous acid, disodium salt Synonyms: Anhydrous sodium sulfite; disodium sulfite; sodium sulphite o 1/ Na · 0 - 8 - 0 · Na Na2S0J MoL. wt: 126.04 Sodium bisulfe Chem. Abstr. Serv Reg. No.: 7631-90-5 Replaced CAS Nos.: 57414-01-4; 69098-86-8; 89830-27-3; 91829-63-9 Chem. Abstr. Name: Sulfurous acid, monosodium salt IUPAC Systematic Name: Sulfurous acid, monosodium salt -131- 132 lARe MONOGRAPHS VOLUME 54 Synonyms: Hydrogen sulfite sodium; monosodium sulfite; sodium acid sulfite; sodium bisulphite; sodium hydrogen sulfite; sodium sulfite (NaHS03) o Il HO - S - a · Na NaHS03 MoL. wt: 104.06 Sodium metabisulfte Chem. Abstr. Serv Reg. No.: 7681-57-4 Altemate CAS No.: 7757-74-6 Replaced CAS No.: 15771-29-6 Chem. Abstr. Name: Disulfurous acid, disodium salt IUPAC Systematic Name: Pyrosulfurous acid, disodium salt Synonyms: Disodium disulfite; disodium metabisulfite; disodium pyrosulfite; sodium disulfite; sodium metabisulphite; sodium pyrosulfite oIl Il0 Na · 0- S - a - S - a · Na .Na2S20S MoL. -
HITTING the MARK in CANCER EPIGENETICS Research in Cancer Epigenetics Is Advancing Rapidly
HITTING THE MARK IN CANCER EPIGENETICS Research in cancer epigenetics is advancing rapidly. Here’s an overview of the tools and methods scientists use to drive progress in the field. For by CUSTOM MEDIA ADVERTISEMENT FEATURE ADVERTISEMENT FEATURE says. “We usually put these cytosine ring — is the best look and behave. Illumina’s Andrew Feber, a cancer patients on a watch and understood epigenetic marker methylation array covers geneticist at UCL Cancer HITTING THE MARK wait scheme. We could have today. Methylation typically 850,000 sites on the Institute in London, is one spared her all that therapy.” silences gene transcription genome, and analyses with of those scientists. He aims Pfister’s experience in patterns that are it, he says, “are sufficient to find epigenetic traces IN CANCER EPIGENETICS illustrates the growing characteristic to specific cell to capture the cancer cell’s of bladder cancer in urine, potential of genomic types and tissues. But when basic identity”. an endeavour he says is RESEARCH IN CANCER EPIGENETICS IS ADVANCING RAPIDLY. Here’s an overview of the tools technologies in epigenetics to it silences tumour suppressor Esteller points out that particularly well suited to and methods scientists use to drive progress in the field. contribute to understanding, genes, cancer may appear. methylation arrays have sequencing since it can diagnosing and treating The first epigenetic many positive aspects, such read the often fragmented cancer. Epigenetic technologies — methylation as low-cost and amenability arrangements of DNA modifications to DNA and arrays — emerged about 15 to formalin-fixed, paraffin- letters in liquid specimens. n 2013 Stefan Pfister, RNA regulate how and when years ago with the launch embedded tissue samples. -
Introduction to Epigenetics Implications for Human Nutrition Outline
Introduction to Epigenetics Implications for Human Nutrition Outline • Epigenetics in Action • Defining (Epi)genetics • ‘Writers’, ‘Readers’ and ‘Erasers’ of epigenetic information • Environmental Epigenomics Nutri(Epi)genomics Epigenetics: “what makes us different even when we are equal” Agouti viable Rainbow yellow Carbon-copy Epigenetics: “what makes us different even when we are equal” - hundreds of different kinds of cells in our bodies - each one derives from the same starting point and has the same 20000 odd genes - as cells develop, their fate is governed by the selective use and silencing of genes - this process is subject to epigenetic factors http://www.ncbi.nlm.nih.gov/About/primer/genetics_cell.html (Epi)genetics • The study of: ‘Any heritable alteration in gene function that does not result in a change in DNA sequence but will have a significant impact on the development of the organism’ The epigenetic marks • DNA Methylation (Me) • Histone modifications (mod) • Non-coding RNAs (ncRNA) www.Epigenome-noe.net/ Epigenetics deals with modifications of chromatin (DNA and associated proteins) which are heritable, reversible and affect genome function (transcription, replication, recombination, chromosome structure, etc.) Why is Epigenetics important? Epigenetics creates a memory of cell identity www.Epigenome-noe.net/ AHEAD Epigenome project (2008) Nature 454: 711 Why is it important to maintain cellular memory? Differentiated cell (e.g.liver cell) + liver cell + liver cell CH3 CH3 CH3 Ac Ac Ac CH3 CH3 CH3 Ac Ac Ac CH3 CH3 CH3 -
In Bisulfite/Permanganate for Organic Compounds Oxidation
Water Research 148 (2019) 198e207 Contents lists available at ScienceDirect Water Research journal homepage: www.elsevier.com/locate/watres New insight into the reactivity of Mn(III) in bisulfite/permanganate for organic compounds oxidation: The catalytic role of bisulfite and oxygen * Shifa Zhong, Huichun Zhang Department of Civil Engineering, Case Western Reserve University, 2104 Adelbert Road, Cleveland, OH, 44106-7201, USA article info abstract À À Article history: A recently discovered bisulfite(HSO3 )/permanganate(MnO4 ) system was reported to produce highly Received 24 June 2018 reactive free Mn(III) that can oxidize organic compounds in milliseconds. However, this characteristic Received in revised form reactivity was not found in all other known reaction systems that can also produce free Mn(III). Why can 18 September 2018 Mn(III) in NaHSO /KMnO be so active? Here, we found NaHSO and O acted as catalysts for the reaction Accepted 12 October 2018 3 4 3 2 between Mn(III) and organic compounds. Without O , 0% of organic compounds were oxidized in Available online 23 October 2018 2 NaHSO3/KMnO4, indicating the absence of O2 inactivated Mn(III) reactivity. When the reaction between NaHSO and KMnO was monitored in air, Mn(III) catalyzed rapid oxidation of NaHSO by O . Then, the Keywords: 3 4 3 2 Bisulfite and permanganate Mn(III) that could oxidize organic compounds was found to be the ones involved in the catalytic reaction Bisulfite/oxygen reaction between NaHSO3 and O2, thus the link between O2 and Mn(III) reactivity was established. Finally, Disproportionation NaHSO3/O2 can be viewed as catalysts for the reaction between Mn(III) and organic compounds because Mn(III) catalyst 1) when Mn(III) was involved in oxidizing organic compounds, it stopped being the catalyst for the Mn(III) oxidant reaction between NaHSO3 and O2 so that they were consumed to a much smaller extent; and 2) without Water treatment NaHSO3 and O2, Mn(III) lost its oxidation ability. -
Winemaking Basics-Bruce Hagen.Pdf
Winemaking Basics Bruce Hagen Sourcing grapes: good wine starts with good grapes Ripeness: is generally expressed as percent sugar or °Brix (°B). The normal range is 22 – 26°B (17.5 to 19 for sparkling and 21 for some ‘crisp’ and austere whites). Use a hydrometer or a refractometer to check it. If you harvest much above 26, you should consider diluting the juice (must) with water to adjust it to downward a bit, depending on the alcohol level you are comfortable with. The problem with making wines from high °Brix grapes is that the resulting alcohol level will be high. The fermentation may stop (stick) and the wine may taste hot. Therefore, you should consider diluting the must or juice, if the sugar level is much above 26 (see adjusting the °Brix below). The alcohol conversion factor for most yeasts is about .57, but ranges from .55 to as high as .64. Multiply the °B by the conversion factor to determine the probable alcohol level: ex 26°B x .57 = 14.8%. If the °B level is 27, the resulting alcohol level will be 15.4 —very hot! If you dilute to 25, the alcohol will be 14.25%. If you dilute it to 24ºB, the alcohol will be 13.7% —quite acceptable. Whites vs. reds: § White grapes are de-stemmed, crushed, and pressed before fermentation. § Skin contact is relatively short. § Red grapes are typically de-stemmed, crushed, cold-soaked (optional), and he wine pressed off the skins and seeds after fermentation. Skin contact is lengthy, so color and tannins are more intense. -
Website Exposure May Cause Bronchitis to Develop with Coughing, ( Or in Your Facility’S RTK Phlegm, And/Or Shortness of Breath
Right to Know Hazardous Substance Fact Sheet Common Name: SODIUM BISULFITE Synonym: Sodium Hydrogen Sulfite CAS Number: 7631-90-5 Chemical Name: Sulfurous Acid, Monosodium Salt RTK Substance Number: 1685 Date: August 1998 Revision: April 2008 DOT Number: UN 2693 (Solution) Description and Use EMERGENCY RESPONDERS >>>> SEE BACK PAGE Sodium Bisulfite is a white, crystalline solid with a slight odor Hazard Summary of rotten eggs. It is often in a liquid solution. It is used in Hazard Rating NJDOH NFPA making paper and leather, as a food preservative and in dye HEALTH 2 - and chemical production. FLAMMABILITY 0 - REACTIVITY 0 - CORROSIVE POISONOUS GASES ARE PRODUCED IN FIRE Reasons for Citation f Sodium Bisulfite is on the Right to Know Hazardous Substance List because it is cited by ACGIH, DOT, NIOSH, Hazard Rating Key: 0=minimal; 1=slight; 2=moderate; 3=serious; 4=severe IARC and EPA. f This chemical is on the Special Health Hazard Substance List. f Sodium Bisulfite can affect you when inhaled. f Contact can severely irritate and burn the skin and eyes. f Inhaling Sodium Bisulfite can irritate the nose, throat and lungs. f Sodium Bisulfite may cause a skin allergy and an asthma- like allergy. SEE GLOSSARY ON PAGE 5. f Sodium Bisulfite is CORROSIVE when in a liquid solution with water. FIRST AID Eye Contact f Quickly brush off excess chemical from the face. Immediately flush with large amounts of water for at least 60 Workplace Exposure Limits minutes, lifting upper and lower lids. Remove contact NIOSH: The recommended airborne exposure limit (REL) is lenses, if worn, while flushing.