Name: methodology for accurate detection of mitochondrial dna methylation
Uploaded: 2018-05-20T13:30:00.000+00:00
Duration: 12 min 11 s
Description: Watch this Scientific Journal Video about Methodology for Accurate Detection of Mitochondrial DNA Methylation at JoVE.com

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><tbody><tr><td>BamHI</td><td>New England BioLabs</td><td># R0136</td><td></td></tr><tr><td>EZ DNA methylation-lightning kit</td><td>Zymo Research</td><td># D5030&amp;nbsp;</td><td></td></tr><tr><td>Qubit ssDNA assay</td><td>Thermo Fisher Scientific</td><td># Q10212</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Thermo Fisher Scientific</td><td># Q32856</td><td></td></tr><tr><td>HotStarTaq plus DNA polymerase kit</td><td>Qiagen</td><td># 203603&amp;nbsp;</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td># 28704</td><td></td></tr><tr><td>NEBNext Ultra DNA Library Kit for Illumina</td><td>New England BioLabs</td><td># E7370S</td><td></td></tr><tr><td>NEBNext Multiplex Oligoes for Illumina</td><td>New England BioLabs</td><td># E7335S</td><td></td></tr><tr><td>AMPure XP Beads</td><td>Beckman Coulter</td><td># A63881</td><td></td></tr><tr><td>High Sensitivity DNA chip</td><td>Agilent</td><td># 5067-4626</td><td></td></tr><tr><td>Qubit high sensitivity dsDNA assay</td><td>Thermo Fisher Scientific</td><td># Q33230</td><td></td></tr><tr><td>MiSeq reagent kit v2 300 cycles</td><td>Illumina</td><td># MS-102-2003</td><td></td></tr><tr><td>PhiX control v3</td><td>Illumina</td><td># FC-110-3001</td><td></td></tr><tr><td>Sodium hydroxide&amp;nbsp;</td><td>Sigma</td><td># S5881</td><td></td></tr><tr><td>Thermal cycler C1000</td><td>Biorad</td><td># 1851148</td><td></td></tr><tr><td>CFX96 Real-Time PCR detection system</td><td>Biorad&amp;nbsp;</td><td>#1855195</td><td></td></tr><tr><td>Qubit Fluorometer</td><td>Thermo Fisher Scientific</td><td># Q33226</td><td></td></tr><tr><td>Bioanalyzer 2100</td><td>Agilent</td><td># G2939BA</td><td></td></tr><tr><td>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 San Sebastian

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

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

甲基化的DNA免疫沉淀

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

科研

生物学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

神经科学

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

生物化学

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦:基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

通过焦磷酸测序对线粒体基因组中的单核苷酸多态性进行基因分型

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

线粒体 DNA 甲基化的精确检测方法

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甲基化的DNA免疫沉淀

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

基于图像的高通量线粒体DNA合成和分布定量

基于图像的高通量线粒体DNA合成和分布定量

基于图像的高通量线粒体DNA合成和分布定量

DNA甲基化：亚硫酸氢修改和分析

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

有针对性的DNA甲基化分析通过新一代测序

通过焦磷酸测序对线粒体基因组中的单核苷酸多态性进行基因分型