德基础代谢研究基金会是在哥本哈根大学的一个独立研究中心, 由来自德基金会的无限制捐赠部分资助。

1. 限制酶治疗
<ol><li>进行线性化线粒体 dna 通过治疗总人类 DNA 与限制酶 BamHI, 那削减在位置14258在人类线粒体脱氧核糖核酸。 注: 对于小鼠 DNA, 使用限制酶 BglII 在相同条件下, 如下所示。</li>
	<li>对于要评估线粒体甲基化的每个样本, 准备一个0.2 毫升反应管, 并添加以下 mix:3 微克基因组 DNA (由荧光量化), 15 ul 缓冲 3, 3 ul BamHI 和水高达 150 ul</li>
	<li>在热循环仪中放置管4小时, 在37摄氏度。</li>
</ol>2. 亚硫酸氢转化
<ol><li>使用亚硫酸氢钠转换 BamHI 处理的 DNA, 如其他地方所述29。</li>
	<li>使用 200-500 ng BamHI 处理的 DNA 为每个转换反应在总容量 20 ul。DNA 回收率为50-70%。</li>
	<li>洗脱 10 ul 洗脱缓冲液中的亚硫酸氢转化 DNA。</li>
	<li>用荧光法定量测定单链 DNA。</li>
	<li>可选: 在继续执行该协议之前, 将亚硫酸氢化转化后的 DNA 储存在-20 摄氏度。长期贮存, 将亚硫酸氢转化后的 DNA 储存在-80 摄氏度</li>
</ol>3. 亚硫酸氢化测序底漆的设计
<ol><li>用于亚硫酸氢的设计底漆使用在线工具 Methprimer30和折半查找 31, 32, 以此进行排序. 注: Methprimer 和折半查找允许搜索 cytosines 转化为 thymines 的基因组序列上的底漆结合序列, 类似于亚硫酸氢后处理。</li>
	<li>为优化和无偏放大, 避免在引物中的 CpG dinucleotides 和设计的扩增子大小之间的 100-300 bp。</li>
	<li>确保设计的底漆是特定的线粒体 DNA 使用折半查找的功能底漆搜索。插入已设计的亚硫酸氢转化底漆, 勾选亚硫酸氢框, 并包括参考基因组和 PCR 参数。 注: 将显示可能的 PCR 产品列表。</li>
	<li>复制前1-5 个底漆序列, 并粘贴到一个排序形式的寡核苷酸合成。 注意: 在实验室中已验证的人和鼠线粒体 dna 底漆序列的列表显示在表 1中。</li>
	<li>根据步骤4中的说明, 采用亚硫酸氢化凝胶电泳法进行的亚硫酸盐转化 PCR 试验底漆。对所有底漆对分别进行测试和优化, 确保琼脂糖凝胶上出现正确的扩增子尺寸。 注: 如果需要复用底漆, 添加多个引物到一个单一的 pcr 反应和可视化 pcr 产品在琼脂糖凝胶电泳或, 更 resolutive 的方法, 如聚丙烯酰胺凝胶电泳, 以区别产品的相似大小。</li>
</ol>4. 亚硫酸氢转化 PCR 和凝胶萃取
<ol><li>为了放大感兴趣的区域, 使用热启动 Taq 聚合酶进行 PCR。简单地, 混合100的 DNA 转换, 500 µM 感和反感底漆, 1 µL dNTP 混合 (每 dNTP 10 µM) 和聚合酶在 7.5 U/反应在总容量50µL. 运行 PCR 以以下情况: 5 分钟在95°c (六十年代94°c; 六十年代55°c *; 六十年代72°c) 35 个周期; <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg">10分钟72°c。单独或复用底漆。</li>
	<li>可选: 当复用引物, 使用 200 ng 转换 DNA 和以下循环条件: 5 分钟95°c (六十年代94°c; 九十年代55°c *; 九十年代72°c) 35 循环; <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg">10分钟72°c。 注: 温度可能因底漆熔化温度而异。</li>
	<li>主题 PCR 产品的凝胶电泳2% 琼脂糖在100伏特。</li>
	<li>在柔和的紫外光下, 用手术刀提取 PCR 产物并加入管内。不要将 PCR 产品暴露在过量的紫外线下, 以避免 DNA 损伤。避免曝光时间超过三十年代。</li>
	<li>使用凝胶纯化方法纯化 PCR 产品, 如前所述3。</li>
	<li>用荧光定量测定步骤4.5 中纯化的 DNA。继续步骤5或在20°c 存储样品。</li>
</ol>5. 亚硫酸氢亚硫酸盐测序库准备
<ol><li>对亚硫酸氢转化 PCR 产物进行库制备。 可选: 在这个阶段多路 PCR 产品。</li>
	<li>使用多达 100 ng 的 PCR 产品进行端修复。添加3µL 端准备酶和6.5 µL 端修复反应缓冲器 (10x) 到55.5 µL PCR 产品在0.2 毫升管。放置和孵化管在热循环仪为30分钟在20°c 之后30分钟在65°c。</li>
	<li>结扎测序适配器通过混合最终修复 DNA 与15µL 钝/TA 连接酶混合, 2.5 µL 适配器和1µL 结扎增强剂。如果使用 < 100 ng PCR 产品作为输入, 稀释适配器10次。</li>
	<li>将混合 (步骤 5.3) 放在热循环仪中, 在20摄氏度孵化15分钟。暂停热循环仪, 并添加3µl 用户酶的管。混合并放置在热循环仪的管, 孵化15分钟在37摄氏度。</li>
	<li>尺寸选择适配器结扎的 dna, 使用固体相可逆固定 (SPRI) 珠的不同比例的珠子, DNA 取决于 PCR 产品大小。</li>
	<li>添加13.5 µL H2O, 从步骤5.4 和55µL 悬浮 SPRI 珠中的适配器结扎 DNA。在室温下孵育5分钟, 在磁性支架上放置管。当溶液清晰时, 将上清液转移到新管, 丢弃含有珠子的管子。 注: 上清包含适配器结扎的 DNA 和大不必要的碎片绑定到丢弃的珠子。</li>
	<li>添加25µL 悬浮 SPRI 珠到上清从步骤 5.6, 混合和孵化5分钟室温。当溶液清晰时, 将管子放在磁性支架上, 并丢弃上清液。 注: 被丢弃的上清液含有不需要的 dna, 而适配器结扎的 dna 则绑定在珠子上。</li>
	<li>当管在磁性立场, 添加200µL 80% 乙醇 (新鲜准备), 以洗涤珠。孵化三十年代, 除去和丢弃上清。重复这一步骤, 共2洗涤。</li>
	<li>空气干珠5分钟, 而管是在磁性立场与一个开放的盖子。</li>
	<li>从磁铁中取出管, 洗脱23µL 洗脱缓冲液中的 DNA。室温孵育2分钟。</li>
	<li>将管子放在磁性支架上, 当溶液清晰时, 将2µL 转移到96井 pcr 板上进行预 pcr 放大 (步骤 5.14)。</li>
	<li>将其余的大约21µL 转移到一个新的0.2 毫升的 PCR 放大管 (步骤 5.17)。 注: 确保不转移任何珠子, 因为珠子可以抑制酶反应的下一步。</li>
	<li>继续步骤5.14 或在20°c 存储样品。</li>
	<li>用 RT qPCR 进行预 PCR 放大, 以估计放大适配器结扎 DNA 所需的周期数。每反应, 添加以下在96井 PCR 板包含2µL DNA (步骤 5.11): 0.4 µL 指数底漆, 0.4 µL 通用 PCR 底漆, 7.2 µL H2O, 10 µL RT-qPCR 主混合。</li>
	<li>将板材置于实时 PCR 机中, 并将板材置于以下情况下: 三十年代在98°c (十年代98°c; 七十五年代65°c) 20 个周期; <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg">5分钟65°c。</li>
	<li>估计每个样本所需的放大周期数, 将一个 ct 值减去与 PCR 反应线性相对应的预放大 RT qPCR 的 ct 值。</li>
	<li>从步骤5.12 中放大的适配器结扎的 dna 混合:21 µL dna, 25 µL pcr 总混合, 1 µL 指数底漆, 1 µL 通用 pcr 底漆和2µL H2O。在热循环仪, 主题每个样品到以下情况: 三十年代在98°c (十年代98°c; 七十五年代65°c) [从步骤 5.16] 周期计算的 Ct; <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg">5分钟65°c。 注意: 请记住, 每个样本只使用一个索引底漆, 以允许多路复用。索引在 PCR 期间入。</li>
	<li>在22µL 洗脱缓冲器中纯化扩增的 DNA SPRI 珠和洗脱。</li>
	<li>采用凝胶电泳或替代方法控制库质量。寻找正确的库峰值大小 (PCR 产品的大小加上适配器)。</li>
	<li>通过涂抹分析估计库产品的平均基对大小: 在高级全局设置中, 在 "涂抹分析" 下双击 "表"。从 100-1000 bp 定义区域, 然后单击确定。在区域表下, 将显示定义的区域, 并计算库的平均大小 (bp)。</li>
	<li>通过荧光量化库。继续步骤6或存储库在-20 °c。</li>
</ol>6. 下一代测序
<ol><li>使用公式计算库的 nM 浓度: <img alt="Equation" src="/files/ftp_upload/57772/57772eq1.jpg">
</li>
	<li>稀释图书馆到同一毫微米浓度例如2毫微米 (选择4毫微米, 2 毫微米, 1 毫微米, 或0.5 毫微米取决于图书馆浓度)。汇集所有库, 以达到 2 nM 库的池。</li>
	<li>变性和稀释图书馆跟随测序仪器指南。简而言之: 变性 2 nM 池添加 0.2 N 氢氧化钠, 在室温下孵育5分钟. 稀释变性池, 最后稀释下午10点。变性和稀释一个商业可利用的控制图书馆到最后稀释 12.5 pM。到新的管, 混合下午10点池与 20% 12.5 pM 控制库。 注: 亚硫酸氢转换引入非常低的复杂性。20% 控制库的峰值将导致更好的排序质量。</li>
	<li>使用深测序仪运行 150 bp 配对端测序。</li>
</ol>7. 计算分析-估计甲基化水平
<ol><li>生成. fastq 文件 (此处称为 sample1_R1.fastq.gz 和 sample1_R2.fastq.gz), 生成所有感兴趣的基因组的俾斯麦索引 (此处位于名为bismarkIndex的文件夹中), 并确保 chrM 的基因组序列可在 fasta 中使用。格式 (此处位于名为chrM的文件夹中)。</li>
	<li>预处理读取以删除引物引入的适配器和偏差: 使用工具 Trim_galore33。从正向和反向读取的 5 ' 端移除两个核苷酸。 trim_galore--clip_R1 2-clip_R2 2-trim1-配对 sample1_R1.fastq.gz sample1_R2.fastq.gz</li>
	<li>使用俾斯麦34将预处理的读取与整个基因组对齐 俾斯麦--燕尾/bismarkIndex/-1/sample1_R1_val_1.fq.gz-2/sample1_R2_val_2.fq.gz 注意: 如果使用了另一个光刻, 请确保丢弃不能唯一对齐的读取。</li>
	<li>提取读取映射到染色体 M, 这里使用Samtools35 samtools 排序-l 0-o sample1_sorted. bam sample1_R1_val_1_bismark_bt2_pe. bam samtools 索引 sample1_sorted. bam samtools 视图-u sample1_sorted. bam chrM |samtools 排序-n o sample1_chrM. bam</li>
	<li>提取甲基化信息 bismark_methylation_extractor-cytosine_report--全面的 genome_folder/chrM/sample1_chrM. bam</li>
	<li>使用. bedGraph、. CX_report 或 .txt 文件进行进一步分析。使用 sample1_chrM.CX_report.txt 文件进行可视化和测试。</li>
	<li>在预处理步骤中执行进一步的裁剪, 如果询问多个区域, 并且在映射过程中生成的 M 偏置图中可以看到底漆偏倚。有关详细信息, 请参阅俾斯麦文档。</li>
</ol>8. 计算分析. 差异检验
<ol><li>确保 CX_report 文件包含7列, 染色体 (这里 chrM), 位置, 链, 甲基化读数的数量, 非甲基化读取的数量, c 上下文和周围序列, 每 C 染色体 m。</li>
	<li>当 CX_report 覆盖 chrM 的整体, 检查子集的区域 (s) 被放大。</li>
	<li>对样本之间的差异使用非参数测试, 例如渔民对单个 C 的确切测试或整个区域的标志测试。 注: 量化通常会接近0% 甲基化, 这就是为什么假设正常不合适的原因。</li>
	<li>同样地, 对于可视化, 可以想象分位数或计算二项比例置信区间。</li>
</ol>

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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. -. 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作者没有竞争的经济利益来申报。

在这里, 我们提供了一种亚硫酸氢测序协议, 专门用于审问线粒体甲基化。与亚硫酸氢酶测序协议用于基因组 DNA 的区别在于利用了先前的限制酵素消化步骤和生物信息学分析, 排除了 NUMT 序列产生的假阳性。
在研究线粒体甲基化时, 我们提供了一种避免亚硫酸氢测序工件的协议。以前报告的亚硫酸氢亚硫酸盐序列工件导致了误报37。蛋白酶 K 去除 DNA 副蛋白的...

线粒体基因组是一个圆形, 双链结构约16.5 公斤的基础 (kb) 长, 构成一个沉重的和轻的链。线粒体基因组存在于每个细胞内的多个拷贝中, 母系遗传, 并编码呼吸链复合体的基本成分1。与细菌基因组相似, 与核基因组不同, 线粒体基因组组织在许多次和三级结构中, 例如在连续和超螺旋结构中 2, 这可以在排序过程中使访问变得困难.实验3。
在细胞核中, DNA 甲基化是一种广泛研究的表观遗传标记, 在许多过程中起着重要作用, 特别是在基因表达调控中。在哺乳动物基因组中, DNA 甲基化主要发生在 deoxycytidines 嘧啶环的5位位置上, 主要是在 CG dinucleotides (或 CpG) 上。胞嘧啶甲基化在体细胞基因组中的70% 都被发现, 占总 DNA 基数的 1%, 为4。DNA methylations 也被描述在非 CpG 的情况下, 如 CpA, CpT 和 CpC, 并存在于各种数量的核 DNA, 其价值高达25% 的甲基化 cytosines 在胚胎干细胞5,6,7。
虽然核基因组的胞嘧啶甲基化被广泛接受, 但线粒体 DNA (线粒体) 甲基的存在仍然存在争议。首次研究线粒体甲基化是在培养细胞中进行的, 在那里线粒体甲基化是容易检测到的, 虽然在较低的水平与核 DNA8。在人类和小鼠细胞中, 线粒体甲基化也被检测到低水平 (2-5%)。使用依赖于5甲基胞嘧啶捕获 (如甲基化 DNA 免疫沉淀 (MeDIP) 和定量 PCR 的检测方法, 在各种鼠标和人和细胞线(9,10 ,) 中也发现线粒体甲基化.11,12。在 ELISA 检测或质谱中使用 5-甲基胞嘧啶抗体, 从纯化的线粒体分数中检测出大量的 DNA 甲基化水平13, 14, 15,16. 然而, 上述研究中的大多数化验方法都使用的技术不是为在单一碱基分辨率下的 DNA 甲基化提供绝对量化的。
定量和 resolutive dna 甲基化分析可以通过一种名为 "亚硫酸氢" 的技术来实现, 利用亚硫酸氢钠的特性将非甲基化胞嘧啶转化为尿的单链 DNA上下文17.利用亚硫酸氢的测序, 一系列研究发现了胞嘧啶甲基化在不同层次的存在。甲基化线粒体在 D 环区域, 12S 或16S 区域在人的18,19,20,21,22,23和老鼠24中容易地被发现。然而, 组织和细胞具有令人着迷的变异, 在研究中总 cytosines 的1-20%。
与这些众多的研究相比, 只有少数研究, 包括从我们的小组, 有争议的存在线粒体甲基化3,25,26,27或质疑的生物学相关性非常低的线粒体水平 (低于 2%)28。最近, 我们报告了一个潜在的亚硫酸氢序列伪影在整个线粒体亚硫酸氢化序列3的观察。我们提供的证据表明, 线粒体 DNA 的二级结构可能导致亚硫酸氢亚硫酸盐测序中的误报, 从而高估甲基化水平。我们这里提供了一个协议, 以防止亚硫酸氢的伪影转换的线粒体。该协议使用简单的酶消化 DNA, 以扰乱线粒体二级结构, 并允许完全进入亚硫酸氢化后, 亚硫酸盐排序协议。此外, 我们还提供了一个伴随的生物信息学管道, 用于分析亚硫酸氢的测序。

<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

利用亚硫酸氢钠的特性, 将非甲基化胞嘧啶转化为尿, 在单链 dna 环境下, 可以实现 dna 甲基化的量化。亚硫酸氢的测序可以是靶向 (使用 PCR) 或在整个基因组进行, 并提供绝对定量的胞嘧啶甲基化的单基分辨率。考虑到细胞核和线粒体 DNA 的独特性质, 特别是在二级结构中, 应该对亚硫酸氢嘧啶测序方法在线粒体中的 adaptions 进行研究。线粒体的二级和三级结构确实可以导致亚硫酸氢的测序工件导致误报, 因为不完全变性, 亚硫酸氢对单链 DNA 的接触较差。在这里, 我们描述了一个协议, 使用酶消化 DNA 与BamHI结合生物信息学分析管道, 以允许准确定量的胞嘧啶甲基化水平在线粒体。此外, 我们还提供了设计特定于线粒体的亚硫酸盐测序底漆的准则, 以避免将不理想的核线粒体片段 (NUMTs) 插入核基因组。

在研究线粒体甲基化时, 该协议的两个步骤至关重要。1) 二次结构的开放性和 2) 线粒体 DNA 特异引物的设计。
通过使用限制酶 BamHI (图 1) 消化人类基因组 dna, 线粒体 dna 结构将在核苷酸位置14258上被切割, 第二结构将被打开。
亚硫酸氢转化的可能损害与完整的?...

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

在这里, 我们提出了一个协议, 允许准确定量的线粒体 DNA (线粒体) 甲基化。在本协议中, 我们描述了一种酶消化 DNA 与BamHI结合的生物信息学分析管道, 可用于避免高估线粒体甲基化水平引起的线粒体二级结构。

线粒体 DNA 甲基化的精确检测方法

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

methodology-for-accurate-detection-of-mitochondrial-dna-methylation

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

13.2K 次观看.  University of Copenhagen. 在这里, 我们提出了一个协议, 允许准确定量的线粒体 DNA (线粒体) 甲基化。在本协议中, 我们描述了一种酶消化 DNA 与BamHI结合的生物信息学分析管道, 可用于避免高估线粒体甲基化水平引起的线粒体二级结构。

视频: Methodology for Accurate Detection of Mitochondrial DNA Methylation

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

使用Cpf1的C-Brick DNA标准装配的方案

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

线粒体 DNA 甲基化的精确检测方法

摘要

探索更多视频

此视频中的章节

为患者组织甲基结合DNA序列捕获

的快速方法新颖的RNA结合蛋白隔离

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

单细胞测序拷贝数改变的检测

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

使用Cpf1的C-Brick DNA标准装配的方案

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

DNA 修饰的免疫: 共焦图像的计算分析

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

全基因组对真核生物转录误差的监测

线粒体 DNA 甲基化的精确检测方法

摘要

探索更多视频

此视频中的章节

为患者组织甲基结合DNA序列捕获

的快速方法新颖的RNA结合蛋白隔离

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

单细胞测序拷贝数改变的检测

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

使用Cpf1的C-Brick DNA标准装配的方案

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

DNA 修饰的免疫: 共焦图像的计算分析

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

全基因组对真核生物转录误差的监测

在果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色