The Novo Nordisk Foundation Centre for Basic Metabolic Research is an independent research center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation.

1. Restriction Enzyme Treatment
<ol>
	<li>Linearize mtDNA by treating total human DNA with the restriction enzyme BamHI, that cuts at position 14258 in the human mitochondrial DNA. 
	NOTE: For mouse DNA, use the restriction enzyme BglII under identical conditions as below.</li>
	<li>For each sample where mtDNA methylation is to be assessed, prepare one 0.2 mL reaction tube and add the following mix:3 &#956;g genomic DNA (quantified by fluorometry), 15 &#956;L Buffer 3, 3 &#956;L BamHI and water up to of 150 &#956;...

<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. R., Fabre, O., Picard, M., 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+Suggesting+Absence+of+Mitochondrial+DNA+Methylation.">Evidence Suggesting Absence of Mitochondrial DNA Methylation.</a> Front Genet. 8, 166 (2017).</li><li>Ehrlich, M., Gama-Sosa, M. 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. 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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. 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The authors have no competing financial interests to declare.

Here, we provide a bisulfite-sequencing protocol which is specifically designed to interrogate mtDNA methylation. The differences with bisulfite-sequencing protocols used for genomic DNA lies in the utilization of a prior restriction enzyme digestion step and a bioinformatic analysis ruling out false positive arising from NUMT sequences.
We provide a protocol to avoid bisulfite-sequencing artifacts when investigating mtDNA methylation. Bisulfite sequencing artifacts leading to false-positives ...

The mitochondrial genome is a circular, double-stranded structure of approximately 16.5-kilo base (kb) long, constituting of a heavy and a light strand. The mitochondrial genome is present in multiple copies within each cell, maternally-inherited, and encodes essential components of the respiratory chain complexes1. Similar to bacterial genomes and unlike the nuclear genome, the mitochondrial genome is organized in numerous secondary and tertiary structures, such as in coiled and supercoiled structures2, which can render access difficult during sequencing experiments3.
In the nucleus, methylation of the DNA is an extensively studied epigenetic mark that plays a role in numerous processes, notably in the regulation of gene expression. In mammalian genomes, DNA methylation occurs primarily on the 5-position of the pyrimidine ring of deoxycytidines, mostly on CG dinucleotides (or CpG). Cytosine methylation is found at 70% of all CpG in the genome of somatic cells and accounts for ~1% of total DNA bases4. DNA methylations has also been described in non-CpG contexts, such as CpA, CpT, and CpC and exist in various amounts in nuclear DNA, with values up to 25% of all methylated cytosines in embryonic stem cells5,6,7.
While cytosine methylation of the nuclear genome is widely accepted, the existence of mitochondrial DNA (mtDNA) methylation is still controversial. The first study investigating mtDNA methylation was performed in cultured cells where mtDNA methylation was readily detected, although at lower levels compared to nuclear DNA8. In both human and murine cells, mtDNA methylation was also detected at low levels (2-5%). Using assays relying on 5 methylcytosine capture such as methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR, mtDNA methylation was also detected in various mouse and human and cells lines9,10,11,12. Using antibodies against 5-methylcytosine in an ELISA assay or mass spectrometry, substantial levels of DNA methylation were detected from purified mitochondrial fractions13,14,15,16. However, most of the assays in the aforementioned studies used techniques that were not designed to provide absolute quantification of DNA methylation at the single base-resolution.
Quantitative and resolutive DNA methylation analysis can be achieved by a technique named &#34;bisulfite sequencing&#34;, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil in single-stranded DNA context17. Using bisulfite sequencing, a constellation of studies has detected the presence of cytosine methylation at various levels. Methylation mtDNA in the D-loop region, the 12S or the 16S region was readily detected in human18,19,20,21,22,23 and mouse24 tissues and cells, however, with an intriguing variability, of 1-20% of total cytosines across studies.
In comparison to these numerous studies, only a few studies, including from our group, have disputed the presence of mtDNA methylation3,25,26,27 or questioned the biological relevance of very low levels of mtDNA levels (below 2%)28. Recently, we reported the observation of a potential bisulfite-sequencing artifact in whole mitochondrial bisulfite sequencing3. We provided evidence that the secondary structure of mitochondrial DNA could lead to false positives in bisulfite sequencing, thereby overestimating methylation levels. We provide here a protocol to prevent an artifact of bisulfite-conversion of mtDNA. This protocol uses a simple enzymatic digestion of DNA to disrupt mtDNA secondary structures and allow full access to bisulfite following a bisulfite sequencing protocol. In addition, we provide an accompanying bioinformatic pipeline for the analysis of bisulfite sequencing.

<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

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Two steps in this protocol are crucial when investigating mtDNA methylation. 1) The opening of the secondary structure and 2) the design of mitochondrial DNA-specific primers.
By digesting the human genomic DNA with the restriction enzyme BamHI (Figure 1), the mitochondrial DNA structure will be cut at nucleotide position 14,258 and the secondary structure will be opened.
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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

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

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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13.1K ビュー.  University of Copenhagen. 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 ...