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 senter for grunnleggende metabolske forskning er en uavhengig forskning ved Universitetet i København delvis finansiert av en ubegrenset donasjon fra Novo Nordisk Foundation.

1. restriksjoner enzym behandling
<ol><li>Linearize mtDNA ved å behandle totale menneskelige DNA med begrensning enzymet BamHI, som kutter sted 14258 i menneskelig Mitokondrielt DNA. NOTE For musen DNA, bruk begrensning enzymet BglII under like idet neden.</li>
	<li>For hver prøve der mtDNA metylering er vurderes, forberede en 0,2 mL reaksjon tube og legge til følgende blanding: 3 μg genomisk DNA (kvantifisert ved fluorometry), 15 μL Buffer 3, 3 μL BamHI og vann opp til 150 μL</li>
	<li>Plassere rør i en ter...

<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. 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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. 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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 gir vi en bisulfite-sekvensering protokollen som er utviklet til å lage mtDNA metylering. Forskjellene med bisulfite-sekvensering protokollsettet for genomisk DNA ligger i bruken av et tidligere begrensning enzym fordøyelsen skritt og en bioinformatic analyse dommen ut falske positive som oppstår fra NUMT sekvenser.
Vi tilbyr en protokoll for å unngå bisulfite-sekvensering gjenstander når undersøke mtDNA metylering. Bisulfite sekvensering gjenstander fører til falske positiver var ...

Mitokondrie genomet er en sirkulær, double-strandet struktur av ca 16,5 kilo base (kb), utgjør en tung og en lys strand. Mitokondrie genomet finnes i flere eksemplarer i hver celle moderskap-arvet, og koder viktige komponenter av respiratoriske kjeden komplekser1. Lik bakteriell genomer og i motsetning til kjernefysiske genomet, mitokondrie genomet er organisert i flere sekundære og tertiære bygninger, som i kveilet og supercoiled2, som kan gjengi tilgang vanskelig under sekvensering eksperimenter3.
I kjernen er metylering av DNA en grundig studert epigenetic merke som spiller en rolle i mange prosesser, spesielt i regulering av genuttrykk. I pattedyr genomer skjer DNA metylering primært på 5-plasseringen av pyrimidine ringen av deoxycytidines, hovedsakelig på CG dinucleotides (eller CpG). Cytosine metylering er funnet på 70% av alle CpG i genomet av somatiske celler og står for ~ 1% av totale DNA baser4. DNA methylations har også blitt beskrevet i ikke-CpG sammenhenger, som CpA, CpT og CpC og finnes i forskjellige mengder i kjernefysiske DNA, med verdier opp til 25% av alle methylated cytosines i embryonale stamceller5,6,7.
Mens cytosine metylering av det kjernefysiske genomet er allment akseptert, er eksistensen av Mitokondrielt DNA (mtDNA) metylering fortsatt kontroversielt. Den første studien undersøker mtDNA metylering ble utført i kulturperler celler der mtDNA metylering ble lett oppdaget, men på lavere nivåer i forhold til kjernefysiske DNA8. I både menneskelig og murine celler, ble mtDNA metylering også funnet på lave nivåer (2-5%). Bruke analyser stole på 5 methylcytosine fange som denaturert DNA immunoprecipitation (MeDIP) etterfulgt av kvantitative PCR, mtDNA metylering ble også funnet i forskjellige mus og menneskelige og celler linjer9,10, 11,12. Bruker antistoffer mot 5-methylcytosine i en ELISA-analysen eller massespektrometri, ble betydelige nivåer av DNA metylering oppdaget renset mitokondrie fraksjoner13,14,15, 16. men mest av analyser i nevnte studiene brukte teknikker som ikke ble utformet for å gi absolutt kvantifisering av DNA metylering i enkelt base-oppløsning.
Kvantitative og resolutive DNA metylering analyse kan oppnås ved en teknikk kalt "bisulfite sekvensering", som utnytter for natrium bisulfite konvertere unmethylated cytosine til uracil i enkelt-strandet DNA sammenheng17 . Bruker bisulfite sekvensering, har en konstellasjon av studier oppdaget tilstedeværelsen av cytosine metylering på ulike nivåer. Metylering mtDNA i regionen D-loop, 12S eller regionen 16S ble lett funnet i menneskelige18,19,20,21,22,23 og mus24 vev og celler, men med en interessant variasjon, 1-20% av totale cytosines på tvers av studier.
I forhold til disse tallrike studier, har bare noen få studier, inkludert fra vår gruppe, omstridt tilstedeværelsen av mtDNA metylering3,25,26,27 eller spurte biologiske relevans svært lave nivåer av mtDNA nivåer (under 2%)28. Nylig rapportert vi observasjon av en potensiell bisulfite-sekvensering gjenstand i hele mitokondrie bisulfite sekvensering3. Vi ga bevis som sekundær strukturen av Mitokondrielt DNA kan føre til falske positiver i bisulfite sekvensering, og dermed overestimating metylering nivåer. Vi gir her en protokoll for å hindre en gjenstand av bisulfite-konvertering av mtDNA. Denne protokollen bruker en enkel enzymatisk fordøyelsen av DNA å avbryte mtDNA sekundære strukturer og gi full tilgang til bisulfite etter en bisulfite sekvensering protokoll. I tillegg gir vi en tilhørende bioinformatic rørledning for analyse av 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

Kvantifisering av DNA metylering oppnås ved hjelp av bisulfite sekvensering, som utnytter for natrium bisulfite konvertere unmethylated cytosine til uracil, i en enkelt-strandet DNA sammenheng. Bisulfite sekvensering kan være målrettet (bruker PCR) eller utført på hele genomet og gir absolutt kvantifisering av cytosine metylering i enkelt base-oppløsning. Gitt forskjellige slags kjernefysisk- og Mitokondrielt DNA, særlig i den sekundære strukturen, bør tilpasninger av bisulfite sekvensering metoder for å undersøke cytosine metylering i mtDNA gjøres. Sekundære og tertiære strukturen i mtDNA kan faktisk føre til bisulfite sekvensering gjenstander fører til falske positiver på grunn av ufullstendige rødsprit dårlig tilgang av bisulfite single-strandet DNA. Her beskriver vi en protokoll med en enzymatisk fordøyelsen av DNA BamHI kombinert med bioinformatic analyse rørledning tillate nøyaktige kvantifisering av cytosine metylering nivåer i mtDNA. Dessuten, vi gir retningslinjer for utforming bisulfite sekvensering primerne gjelder for mtDNA, for å unngå målretting uønskede kjernefysiske mitokondrie segmenter (NUMTs) inn kjernefysiske genomet.

To trinnene i denne protokollen er viktig når undersøke mtDNA metylering. 1) åpningen av sekundær strukturen og 2) utformingen av Mitokondrielt DNA-spesifikke primere.
Ved fordøye menneskelige genomisk DNA med begrensning enzymet BamHI (figur 1), Mitokondrielt DNA strukturen vil bli kuttet nukleotid posisjonen 14,258 og sekundære strukturen vil bli åpnet.
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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

Her presenterer vi en protokoll for å tillate nøyaktige kvantifisering av Mitokondrielt DNA (mtDNA) metylering. I denne protokollen beskriver vi en enzymatisk fordøyelsen av DNA med BamHI kombinert med en bioinformatic analyse rørledning hvilke kan brukes å unngå overvurdering av mtDNA metylering nivåer forårsaket av sekundære strukturen i mtDNA.

Metodikk for nøyaktig påvisning av Mitokondrielt DNA metylering

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

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