Name: methodology for accurate detection of mitochondrial dna methylation
Uploaded: 2018-05-20T13:30:00
Description: Watch this Scientific Journal Video about Methodology for Accurate Detection of Mitochondrial DNA Methylation at JoVE.com

Novo Nordisk Foundation Center for metaboliske grundforskning er en uafhængig Forskningscenter på Københavns Universitet delvist finansieret af en ubegrænset donation fra Novo Nordisk Fonden.

1. begrænsning enzym behandling
<ol><li>Linearize mtDNA ved at behandle samlede menneskelige DNA med begrænsning enzymet BamHI, der skærer på position 14258 i den menneskelige mitokondrie-DNA. Bemærk: For musen DNA, bruge begrænsning enzymet BglII under identiske forhold som nedenfor.</li>
	<li>For hver-prøve, hvor mtDNA methylering er at blive vurderet, forberede en 0,2 mL reaktion tube og tilføje den følgende blanding: 3 μg genomisk DNA (kvantificeres ved fluorometry), 15 μl Buffer 3, 3 μL BamHI og van...

<ol>
	<li>Falkenberg, M., Larsson, N. -. G., Gustafsson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+and+transcription+in+mammalian+mitochondria.">DNA replication and transcription in mammalian mitochondria.</a> Annu Rev Biochem. 76, 679-699 (2007).</li><li>Kolesar, J. E., Wang, C. Y., Taguchi, Y. V., Chou, S. -. H., Kaufman, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+intact+mitochondrial+DNA+agarose+electrophoresis+reveals+the+structural+complexity+of+the+mammalian+mitochondrial+genome.">Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome.</a> Nucleic Acids Res. 41 (4), e58 (2013).</li><li>Mechta, M., Ingerslev, L. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amount+and+distribution+of+5-methylcytosine+in+human+DNA+from+different+types+of+tissues+of+cells.">Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.</a> Nucleic Acids Res. 10 (8), 2709-2721 (1982).</li><li>Lister, R., Pelizzola, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462 (7271), 315-322 (2009).</li><li>Yan, J., Zierath, J. R., Barres, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+non-CpG+methylation+in+mammals.">Evidence for non-CpG methylation in mammals.</a> Exp Cell Res. 317 (18), 2555-2561 (2011).</li><li>Patil, V., Ward, R. L., Hesson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evidence+for+functional+non-CpG+methylation+in+mammalian+cells.">The evidence for functional non-CpG methylation in mammalian cells.</a> Epigenetics. 9 (6), 823-828 (2014).</li><li>Nass, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+methylation+of+mitochondrial+and+nuclear+DNA+in+cultured+mouse,+hamser+and+virus+trasnforemd+hamser+cells+in+vivo+and+in+vitro+methylation.">Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamser and virus trasnforemd hamser cells in vivo and in vitro methylation.</a> J Mol Biol. 80 (1), 155-175 (1973).</li><li>Shock, L. S., Thakkar, P. V., Peterson, E. J., Moran, R. G., Taylor, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methyltransferase+1,+cytosine+methylation,+and+cytosine+hydroxymethylation+in+mammalian+mitochondria.">DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria.</a> PNAS. 108 (9), 3630-3635 (2011).</li><li>Bellizzi, D., D'Aquila, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Control+Region+of+Mitochondrial+DNA+Shows+an+Unusual+CpG+and+Non-CpG+Methylation+Pattern.">The Control Region of Mitochondrial DNA Shows an Unusual CpG and Non-CpG Methylation Pattern.</a> DNA Res. 20 (6), 537-547 (2013).</li><li>Jia, Y., Li, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Low-Protein+Diet+Affects+Epigenetic+Regulation+of+Hepatic+Mitochondrial+DNA+Transcription+in+a+Sex-Specific+Manner+in+Newborn+Piglets+Associated+with+GR+Binding+to+Its+Promoter.">Maternal Low-Protein Diet Affects Epigenetic Regulation of Hepatic Mitochondrial DNA Transcription in a Sex-Specific Manner in Newborn Piglets Associated with GR Binding to Its Promoter.</a> PLoS ONE. 8 (5), e63855 (2013).</li><li>Jia, Y., Song, H., Gao, G., Cai, D., Yang, X., Zhao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Betaine+Supplementation+during+Gestation+Enhances+Expression+of+mtDNA-Encoded+Genes+through+D-Loop+DNA+Hypomethylation+in+the+Skeletal+Muscle+of+Newborn+Piglets.">Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.</a> J Agric Food Chem. 63 (46), 10152-10160 (2015).</li><li>Infantino, V., Castegna, A., Iacobazzi, F., Spera, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+methyl+cycle+affects+mitochondrial+methyl+availability+and+glutathione+level+in+Down's+syndrome.">Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome.</a> Mol Genet Metab. 102 (3), 378-382 (2011).</li><li>Chen, H., Dzitoyeva, S., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+valproic+acid+on+mitochondrial+epigenetics.">Effect of valproic acid on mitochondrial epigenetics.</a> Eur J Pharmacol. 690 (1-3), 51-59 (2012).</li><li>Dzitoyeva, S., Chen, H., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aging+on+5-hydroxymethylcytosine+in+brain+mitochondria.">Effect of aging on 5-hydroxymethylcytosine in brain mitochondria.</a> Neurobiol Aging. 33 (12), 2881-2891 (2012).</li><li>Menga, A., Palmieri, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLC25A26+overexpression+impairs+cell+function+via+mtDNA+hypermethylation+and+rewiring+of+methyl+metabolism.">SLC25A26 overexpression impairs cell function via mtDNA hypermethylation and rewiring of methyl metabolism.</a> FEBS J. 284 (6), 967-984 (2017).</li><li>Frommer, M., McDonald, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> PNAS. 89 (5), 1827-1831 (1992).</li><li>Byun, H. -. M., Panni, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+airborne+pollutants+on+mitochondrial+DNA+Methylation.">Effects of airborne pollutants on mitochondrial DNA Methylation.</a> Part Fibre Toxicol. 10 (1), 1 (2013).</li><li>Janssen, B. G., Byun, H. -. M., Gyselaers, W., Lefebvre, W., Baccarelli, A. A., Nawrot, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+mitochondrial+methylation+and+exposure+to+airborne+particulate+matter+in+the+early+life+environment:+An+ENVIRONAGE+birth+cohort+study.">Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study.</a> Epigenetics. 10 (6), 536-544 (2015).</li><li>Zheng, L. D., Linarelli, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+is+associated+with+epigenetic+and+genetic+regulation+of+mitochondrial+DNA+in+obese+humans.">Insulin resistance is associated with epigenetic and genetic regulation of mitochondrial DNA in obese humans.</a> Clin Epigenetics. 7 (1), 1739 (2015).</li><li>Byun, H. -. M., Colicino, E., Trevisi, L., Fan, T., Christiani, D. C., Baccarelli, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Air+Pollution+and+Blood+Mitochondrial+DNA+Methylation+on+Markers+of+Heart+Rate+Variability.">Effects of Air Pollution and Blood Mitochondrial DNA Methylation on Markers of Heart Rate Variability.</a> J Am Heart Assoc. 5 (4), (2016).</li><li>Bianchessi, V., Vinci, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrion.">Mitochondrion.</a> MITOCH. 27 (C), 40-47 (2016).</li><li>Wijst, M. G. P., van Tilburg, A. Y., Ruiters, M. H. J., Rots, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mitochondria-targeted+DNA+methylation+identifies+GpC+methylation,+not+CpG+methylation,+as+potential+regulator+of+mitochondrial+gene+expression.">Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression.</a> Sci Rep. 7 (1), 177 (2017).</li><li>Wong, M., Gertz, B., Chestnut, B. A., Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNMT3A+and+DNA+methylation+in+skeletal+muscle+and+CNS+of+transgenic+mouse+models+of+ALS.">Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS.</a> Front cellr Neurosci. 7, 279 (2013).</li><li>Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-methylcytidylic+acid:+absence+from+mitochondrial+DNA+of+frogs+and+HeLa+cells.">5-methylcytidylic acid: absence from mitochondrial DNA of frogs and HeLa cells.</a> Science. 184 (4132), 80-81 (1974).</li><li>Gama-Sosa, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Levels and distribution of 5-methylcytosine in Chordate DNA. , 1-201 (1985).</li><li>Hong, E. E., Okitsu, C. Y., Smith, A. D., Hsieh, C. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionally+specific+and+genome-wide+analyses+conclusively+demonstrate+the+absence+of+CpG+methylation+in+human+mitochondrial+DNA.">Regionally specific and genome-wide analyses conclusively demonstrate the absence of CpG methylation in human mitochondrial DNA.</a> Mol Cell Biol. 33 (14), 2683-2690 (2013).</li><li>Liu, B., Du, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpG+methylation+patterns+ofhuman+mitochondrial+DNA.">CpG methylation patterns ofhuman mitochondrial DNA.</a> Nature Publishing Group. , 1-10 (2016).</li><li>Donkin, I., Versteyhe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+and+Bariatric+Surgery+Drive+Epigenetic+Variation+of+Spermatozoa+in+Humans.">Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans.</a> Cell Metab. 23 (2), 369-378 (2016).</li><li>Li, L. C., Dahiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MethPrimer:+designing+primers+for+methylation+PCRs.">MethPrimer: designing primers for methylation PCRs.</a> Bioinformatics. 18 (11), 1427-1431 (2002).</li><li>Tusn&#225;dy, G. E., Simon, I., V&#225;radi, A., Ar&#225;nyi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiSearch:+primer-design+and+search+tool+for+PCR+on+bisulfite-treated+genomes.">BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes.</a> Nucleic Acids Res. 33 (1), e9 (2005).</li><li>Ar&#225;nyi, T., V&#225;radi, A., Simon, I., Tusn&#225;dy, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BiSearch+web+server.">The BiSearch web server.</a> BMC bioinformatics. 7, 431 (2006).</li><li>Krueger, F., Andrews, S. 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Her give vi et bisulfite-sekventering protokol, som er specielt designet til at afhøre mtDNA methylering. Forskelle med bisulfite-sekventering protokoller, der bruges for genomisk DNA ligger i udnyttelsen af en forudgående begrænsning enzym fordøjelsen skridt og en bioinformatic analyse dom ud falsk positive foelger NUMT sekvenser.
Vi leverer en protokol for at undgå bisulfite-sekventering artefakter, når undersøger mtDNA methylering. Bisulfite sekventering artefakter fører til falsk-p...

Den mitokondrielle genom er en cirkulær, dobbelt-strenget struktur af cirka 16,5-kilo base (kb) længe, som udgør en tung og en let strand. Den mitokondrielle genom er til stede i flere eksemplarer i hver celle, maternally-arvet, og koder væsentlige komponenter af respiratorisk kæde komplekser1. Svarende til bakteriel genomer og i modsætning til den nukleare genom, den mitokondrielle genom er organiseret i adskillige sekundære og tertiære strukturer, såsom i snoede og supercoiled strukturer2, der kan gøre adgang vanskeligt under sekventering forsøg3.
I kernen er methylering af DNA et grundigt studeret epigenetiske mark, der spiller en rolle i mange processer, navnlig reguleringen af genekspression. I pattedyr genomer forekommer DNA methylering primært på 5-position af pyrimidin ring af deoxycytidines, for det meste på CG dinucleotides (eller CpG). Cytosin methylering er fundet på 70% af alle CpG i genomet af somatiske celler og tegner sig for ~ 1% af den samlede DNA baserer4. DNA methylations er også blevet beskrevet i ikke-CpG sammenhænge, såsom CpA, CpT og CpC og findes i forskellige mængder i nukleare DNA med værdier op til 25% af alle methylerede cytosines i embryonale stamceller5,6,7.
Mens cytosin methylering af det nukleare genom er bredt accepteret, er eksistensen af mitokondriel DNA (mtDNA) methylering stadig kontroversielt. Den første undersøgelse behandlende mtDNA methylering blev udført i kulturperler celler hvor mtDNA methylering blev let fundet, selv om på lavere niveauer i forhold til nukleare DNA8. I både menneskelige og murine celler, blev mtDNA methylering også fundet på et lavt niveau (2-5%). Ved hjælp af assays afhængige 5 methylcytosine fange såsom methylerede DNA immunoprecipitation (MeDIP) efterfulgt af kvantitativ PCR mtDNA methylering blev også fundet i forskellige mus og menneskelige og celler linjer9,10, 11,12. Ved hjælp af antistoffer mod 5-methylcytosine i en ELISA assay eller massespektrometri, blev betydelige niveauer af DNA methylering opdaget fra renset mitokondrie fraktioner13,14,15, 16. men de fleste af assays i de ovennævnte undersøgelser anvendes teknikker, der ikke var designet til at give absolut kvantificering af DNA methylering i den fælles base-opløsning.
Kvantitative og resolutive DNA methylering analyse kan opnås ved en teknik kaldet "bisulfite sekventering", som tager fordel af natrium bisulfite til at konvertere unmethylated cytosin til uracil i enkeltstrenget DNA sammenhæng17 ejendom . Bruger bisulfite sekventering, har en konstellation af undersøgelser konstateret tilstedeværelsen af cytosin methylering på forskellige niveauer. Methylering mtDNA i D-loop region, 12S eller regionen 16S blev let fundet i menneskelige18,19,20,21,22,23 og mus24 væv og celler, dog med en spændende variation, 1-20% af total cytosines på tværs af undersøgelser.
I forhold til disse talrige undersøgelser, har kun et par undersøgelser, herunder fra vores gruppe, bestridt tilstedeværelsen af mtDNA methylering3,25,26,27 eller spørgsmålstegn ved biologisk relevans meget lave niveauer af mtDNA niveauer (under 2%)28. For nylig, vi rapporterede observation af en potentiel bisulfite-sekventering artefakt i hele mitokondrie bisulfite sekventering3. Vi fremlagde beviser, at den sekundære struktur af mitokondrie-DNA kan føre til falske positiver i bisulfite sekventering, dermed overvurderer methylering niveauer. Vi giver her en protokol for at forhindre en artefakt af bisulfite-konvertering af mtDNA. Denne protokol bruger en simpel Enzymatisk nedbrydning af DNA til at forstyrre mtDNA sekundære strukturer og give fuld adgang til bisulfite efter en bisulfite sekventering protokol. Derudover giver vi en ledsagende bioinformatic rørledning til analyse af bisulfite sekvensering.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">BamHI</td>
			<td style="width:164px;">New England BioLabs</td>
			<td style="width:113px;"># R0136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ DNA methylation-lightning kit</td>
			<td>Zymo Research</td>
			<td># D5030&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit ssDNA assay</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q10212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit assay tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStarTaq plus DNA polymerase kit</td>
			<td>Qiagen</td>
			<td># 203603&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td># 28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEBNext Ultra DNA Library Kit for Illumina</td>
			<td>New England BioLabs</td>
			<td># E7370S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEBNext Multiplex Oligoes for Illumina</td>
			<td>New England BioLabs</td>
			<td># E7335S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td># A63881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Sensitivity DNA chip</td>
			<td>Agilent</td>
			<td># 5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit high sensitivity dsDNA assay</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q33230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiSeq reagent kit v2 300 cycles</td>
			<td>Illumina</td>
			<td># MS-102-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PhiX control v3</td>
			<td>Illumina</td>
			<td># FC-110-3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide&#160;</td>
			<td>Sigma</td>
			<td># S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal cycler C1000</td>
			<td>Biorad</td>
			<td># 1851148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX96 Real-Time PCR detection system</td>
			<td>Biorad&#160;</td>
			<td>#1855195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q33226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td># G2939BA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiSeq instrument</td>
			<td>Illumina</td>
			<td># SY-410-1003</td>
			<td></td>
		</tr>
	</tbody>
</table>

methodology for accurate detection of mitochondrial dna methylation

Kvantificering af DNA methylering kan opnås ved hjælp af bisulfite sekventering, som tager fordel af natrium bisulfite til at konvertere unmethylated cytosin til uracil, i en enkelt-strenget DNA sammenhæng ejendom. Bisulfite sekvensering kan være målrettet (ved hjælp af PCR) eller udføres på den samlede genom og giver absolut kvantificering af cytosin methylering i den fælles base-opløsning. Givet den særskilte karakter af nukleare og mitokondrie DNA, navnlig i den sekundære struktur, bør tilpasninger af bisulfite sekventering metoder for at undersøge cytosin methylering i mtDNA gøres. Sekundære og tertiære struktur af mtDNA kan faktisk føre til bisulfite sekventering artefakter fører til falsk-positive på grund af ufuldstændige denaturering ringe adgang for bisulfite til enkeltstrenget DNA. Her, beskriver vi en protokol med en Enzymatisk nedbrydning af DNA med BamHI kombineret med bioinformatic analyse pipeline tillade nøjagtig kvantificering af cytosin methylering niveauer i mtDNA. Desuden vi give retningslinjer til at designe bisulfite sekventering primere specifikke for mtDNA, for at undgå rettet mod uønskede nukleare mitokondrie segmenter (NUMTs) indsat i det nukleare genom.

To trin i denne protokol er afgørende når undersøger mtDNA methylering. 1) åbningen af sekundær struktur og 2) udformningen af mitokondriel DNA-specifikke primere.
Fordøje det humant genomisk DNA med begrænsning enzymet BamHI (figur 1), den mitokondrielle DNA struktur vil blive skåret på nukleotid position 14,258 og sekundær struktur åbnes.
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Watch this Scientific Journal Video about Methodology for Accurate Detection of Mitochondrial DNA Methylation at JoVE.com

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Vi præsenterer her, en protokol til at lade nøjagtig kvantificering af mitokondriel DNA (mtDNA) methylering. I denne protokol beskriver vi en Enzymatisk nedbrydning af DNA med BamHI kombineret med en bioinformatic analyse pipeline, som kan bruges til at undgå overvurdering af mtDNA methylering niveauer forårsaget af mtDNA sekundær struktur.

Metoder til nøjagtig detektering af mitokondrie-DNA methylering

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

The overall goal of this bisulfite sequencing protocol and bioinformatic pipeline is to achieve an accurate quantification of mitochondrial DNA methylation. This method can help answer key questions in the mitoepigenetic field such as is mitochondrial DNA methylation changing human disease? The main advantage of this technique is that it allows for accurate quantification of mitochondrial DNA methylation.Crucial step when investigating mitochondrial DNA methylation is to linearize the mitochondrial DNA by BamH1 digestion before treatment with sodium bisulfite. The BamH1 restriction enzyme site is displayed in this map of the human mitochondrial genome. Also shown are the five regions of the mitochondrial genome that will be investigated by bisulfite sequencing.Displacement loop, tRNA phenylalanine, and 12S ribosome, 16S ribosome, NADH dehydrogenase 5, cytochrome B mRNA encoding gene. After designing the primers for bisulfite sequencing, using online tools as described in the text protocol, primer specificity toward the mitochondrial genome is verified using BiSearch. Primer sequences are added to the BiSearch function primer search.The bisulfite box is checked and a reference genome is selected. The search results will show potential PCR products generated on sense and antisense chains. To amplify the regions of interest from the bisulfite converted mitochondrial genome, perform PCR using a Hot-start Taq polymerase.If using primers separately, mix 100 nanograms of converted DNA, 500 micromolar of sense and antisense primers, one microliter dNTP mix and polymerase at 7.5 units per reaction in a total volume of 50 microliters. Run the PCR with the following conditions:five minutes at 95 degrees Celsius, one minute at 94 degrees Celsius, one minute at 55 degrees Celsius, one minute at 72 degrees Celsius for 35 cycles, followed by 10 minutes at 72 degrees Celsius. For multiplexing primers, use 200 nanograms of converted DNA in the following cycling conditions:five minutes at 95 degrees Celsius, one minute at 94 degrees Celsius, 1.5 minutes at 55 degrees Celsius, and 1.5 minutes at 72 degrees Celsius for 35 cycles, followed by 10 minutes at 72 degrees Celsius.When the PCR is complete, perform gel electrophoresis on the PCR products, using a 2%agarose at 100 volts. Extraction of the PCR products from the agarose gel must be done in a way that minimizes UV exposure and the risk of DNA damage. Take a photo of the gel under UV light.Then, very quickly, mark around the PCR product with a scalpel and turn off the UV light. Next, cut the PCR product slowly without exposure to UV light. Place the gel slices in microcentrifuge tubes and purify the PCR products using gel purification as described in the text protocol.Quantify the purified DNA using fluorometry. Store the samples at 20 degrees Celsius, if not proceeding immediately to bisulfite sequencing library preparation. Begin the library preparation of bisulfite converted PCR products by performing end repair using up to 100 nanograms of PCR product.To each 0.2 milliliter tube, add 55.5 microliters of PCR product, three microliters of end prep enzyme, and 6.5 microliters of 10X end repair reaction buffer. Incubate the tubes in a thermocycler at 20 degrees Celsius for 30 minutes, followed by 65 degrees Celsius for 30 minutes. Next, ligate sequencing adapters by mixing the end repaired DNA with 15 microliters of Blunt/TA ligase mix, 2.5 microliters of adapter, and one microliter of ligation enhancer.Place the mix in a thermocycler, and incubate at 20 degrees Celsius for 15 minutes. Pause the thermocycler and add 3 microliters of USER enzyme to each tube. Mix and place the tubes back in the thermocycler and incubate at 37 degrees Celsius for 15 minutes.Size select the adaptor-ligated DNA using solid phase reversible immobilization or SPRI. Add to the adaptor-ligated DNA 13.5 microliters of water and 55 microliters of resuspended SPRI beads. Incubate at room temperature for five minutes.Place the tubes on a magnetic stand. When the solution is clear, transfer the supernatant, containing the adaptor-ligated DNA, to a new tube, and discard the tube containing the beads. Add 25 microliters of resuspended SPRI beads to the supernatant;mix and incubate at room temperature for five minutes.Place the tubes on the magnetic stand and when the solution is clear, discard the supernatant. The adaptor-ligated DNA is now bound to the beads. While the tubes are on the magnetic stand, add 200 microliters of freshly prepared 80%ethanol to each tube and incubate for 30 seconds to wash the beads.Remove and discard the supernatant. Repeat this ethanol wash step for a total of two washes. Remove the ethanol from the second wash and air dry the beads by leaving the tubes on the magnetic stand, with the lid open, for five minutes.After five minutes, remove the tubes from the magnetic stand, add 23 microliter of DNA elution buffer to the beads and mix. Incubate at room temperature for two minutes. Place the tubes on the magnetic stand until the solution is clear.Then, transfer two microliter of the solution to a 96-Well PCR plate for the pre-PCR amplification. While carefully avoiding the beads, transfer the rest of the solution to a new 0.2 milliliter tube for PCR amplification. Perform pre-PCR amplification by RT-qPCR to estimate the number of cycles needed to amplify the adaptor-ligated DNA.Make a master mix of the following per reaction used. 0.4 microliters of index primer, 0.4 microliters of universal PCR primer, 7.2 microliters of water, and 10 microliters of RT-qPCR master mix. Then, mix the master mix and add 18 microliters of master mix to the two microliters of DNA in the 96-Well PCR plate.Place the plate in a real-time PCR machine and perform amplification under the following conditions:30 seconds at 98 degrees Celsius, 10 seconds at 98 degrees Celsius, and 75 seconds at 65 degrees Celsius for 20 cycles, followed by five minutes at 65 degrees Celsius. After estimating the number of amplification cycles needed for each sample, as described in the text protocol, amplify the adaptor-ligated DNA. For each sample, mix 21 microliters of DNA, 25 microliters of PCR master mix, one microliter of index primer, one microliter of universal PCR primer, and two microliters of water.In the thermocycler, subject each sample to the following conditions:30 seconds at 98 degrees Celsius, 10 seconds at 98 degrees Celsius, and 75 seconds at 65 degrees Celsius for the calculated number of cycles, followed by five minutes at 65 degrees Celsius. Purify the amplified DNA using SPRI beads, as demonstrated earlier and elute in 22 microliters of elution buffer. Control the library quality by using a Bioanalyzer.Look for correct library peak sizes. Estimate the average base pair size of the library products by smear analysis. In Advanced Global Settings, under Smear Analysis, double-click on Table.Find the region from 100 to 1000 base pair and click OK.Under Region Table, the defined region will appear and the library average size is calculated. Quantify the library by fluorometry. Store the library at 20 degrees Celsius until ready for next generation sequencing and computational analyses.By bisulfite sequencing, mitochondrial DNA methylation was investigated in undigested and digested total DNA from human skeletal muscle cells at five different regions of the mitochondrial genome. A full square represents cytosine guanine dinucleotide sites. The open square represents non-cytosine guanine dinucleotide sites.For undigested mitochondrial DNA, methylation levels ranged from 0-15.1%across all investigated regions. However, when the DNA was digested prior to bisulfite conversion, the uncoversion rate dropped to a maximum of 1%across all regions. This illustrates the importance of BamH1 digestion prior to bisulfite conversion to avoid overestimation of mitochondrial DNA methylation levels.After watching this video, you should have a good understanding on how to perform mitochondrial bisulfite sequencing, and by following our bioinformatic pipeline, you can achieve accurate quantification of mitochondrial DNA methylation.

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