这项工作是由乔治亚癌症联盟授予- R9028支持。

理由利用使用的Thermo Scientific Dharmacon寡核苷酸，50到80个碱基，在这里我们提出一个过程，基于一个基因校正实验，寡核苷酸可以遗传信息转移到酵母细胞的基因组DNA后，退火目标染色体DNA。通过寡核苷酸的成功目标是打进显示预期的表型酵母殖民地的外观。如果所需的基因改造进行在寡核苷酸序列中的一个或多个核苷酸，遗传信息流直接从RNA道染色体的目标DNA，因此，在染色体水平在定位过程中的RNA / DNA杂交的形式。一个的Thermo Scientific Dharmacon的RNA含寡核苷酸/ S可以作为模板，通过DNA修复合成基因打靶或可以嵌入到DNA为模板，在DNA复制服务。在这些实验中，我们使用的酵母/酵母/真核细胞模型系统，然而，类似的方法还可以应用在其他生物体或细胞类型。在这个视频中，我们使用热科学Dharmacon寡核苷酸的同源性设计目标酵母的基因组位点，并包含在中间的一个核苷酸，以正确的靶基因的突变。改造后的RNA含有寡，我们观察到目标站点的修正，在一定的频率，这表明，道寡核苷酸的RNA可以纳入染色体DNA作为DNA合成的模板，在DNA复制。内的RNA道进行稳定的遗传信息传送到下面的单元格几代人。在这里，我们描述了酵母细胞转化的Thermo Scientific Dharmacon RNA含寡核苷酸和方法步骤，以检测RNA信息转移到染色体DNA。 答：在本实验中使用的RNA含寡糖的设计我们设计的一种RNA寡核苷酸，纠正在酵母的遗传缺陷s 酵母染色体DNA trp5轨迹。在协议中使用的酵母菌株包含一个突变 trp5基因两碱基缺失和一个无义突变（图1A） 。这种酵母是一种色氨酸营养缺陷型突变体，并不构成没有色氨酸媒体上的殖民地。在酵母基因组数据库（http://www.yeastgenome.org/）中的TRP5基因的DNA序列是可用的。的Thermo Scientific Dharmacon RNA含寡核苷酸在本议定书中使用一个65 - MER与DNA插入一个2基地，旨在纠正的缺失突变，旨在纠正 trp5等位基因（图1A）无义突变的核苷酸。寡核苷酸序列设计如下： 5&#39; - AAAAGGGTTTTGATGAAGCTGTCG - CG - GATCCCACATTCTG - RG - GAAGACTTCAAATCCTTGTATTCT。这是合成寡核苷酸的Thermo Scientific Dharmacon（拉斐特，CO）在200纳米尺度，是脱盐，deprotected没有页面净化使用。 B.制备酵母转化RNA含有寡糖（Storici 等修改，2007年1） <ol><li>擦拭将用于实验，包括寡管，移液器，旋涡，衣架，实验区，并与核糖核酸酶净化解决方案，以消除潜在的RNase污染的一切开始之前研究员戴手套的材料。使用无RNase水，化学试剂，试管和枪头在所有步骤。在这个实验中的每一个步骤应该是无RNase。 </li><li>重悬的Thermo Scientific Dharmacon RNA含有寡收到该公司以250 pmoles /μL无RNase的水涡大力溶解沉淀原液。储存在-80 ° C。 </li><li>改造前，立即在冰上解冻的RNA寡核苷酸和无RNase在无RNase管的水稀释50 pmoles /μL。每个转换需要1 nmole含有RNA的寡核苷酸。 </li><li>变性的选择金额在100℃2分钟加热块含有RNA的寡核苷酸，以消除对寡核苷酸二级结构。 </li><li>变性后立即放置在冰上管。在冰上保持到转型。 </li><li>在实验中控制了相应的DNA只寡核苷酸用于。这寡核苷酸是从-20 ° C解冻，并转变为寡核苷酸的RNA含准备，如上面所述。 </li></ol> C.酵母细胞的转化，使用RNA含有寡糖<ol><li> trp5突变的酵母细胞接种5毫升丰富的YPD液体培养基中，成长在30 ° C过夜（见材料）。 </li><li>转移到YPD液体培养基中50毫升1.5毫升的隔夜文化。 </li><li>在30℃摇床（225转）4H中孵育细胞。 </li><li>准备解决方案1紧接在无RNase管改造和解决方案2（见材料）。 </li><li>将细胞培养50毫升无RNase管，并在3000转，对应1562年克的2分钟，旋转。细胞沉淀的沉淀约。 0.3厘米3。 </li><li>取出50毫升无RNase水，并在2分钟3000转的旋转上清，洗涤细胞。 </li><li> 5次重复第6步，培养基和核糖核酸酶，可以在中尽可能摆脱。 </li><li>取出5毫升的解决方案1和2分钟的转速在3000旋转上清，悬浮细胞。 </li><li>删除的解决方案1 ​​250μL上清和悬浮细胞。这种细胞的数量是足够7-8转换。 </li><li>分装50μL细胞悬液，在无RNase离心管，加入20μl的RNA含寡工作的解决方案（1 nmole），或20μL的DNA寡核苷酸工作的解决方案（1 nmole），或20μL无菌水没有为阴性对照寡核苷酸。每个转化反应，然后加入300μL的解决方案2。有没有需要添加在转型过程中的鲑鱼精子DNA，寡核苷酸为载体本身的行为。 </li><li>涡大力混合元件均匀。 </li><li> 30 ° C时30分在摇床孵育转化反应。 </li><li>热休克在42 ° C为15分钟。 </li><li>在5000转，相当于2236克4分钟，离心细胞。 </li><li>取出100μL无菌水上清和悬浮细胞。 </li><li>这个细胞悬液等分，并用无菌水稀释十万倍和YPD使用约板的板细胞。 15无菌玻璃珠和孵化在30 ° C时2天。 </li><li>板从每改造一个合成色氨酸（SC -色氨酸）使用约不完整的固体培养基的Petri菜反应中的所有重悬细胞。 15无菌玻璃珠和孵化在30 ° C时4-5天（图2）。 </li></ol> D.分析RNA含寡糖基因校正<ol><li>计数的选择性培养基（图2），以及YPD培养基上生长的菌落计算的RNA寡核苷酸基因校正频率，唯一的DNA寡核苷酸和无寡控制数量。比较后所得的数字。 trp5两个碱基的缺失和无义突变的等位基因的自发回归率小于10-9使用的酵母菌株，因此，我们期望寡核苷酸时没有添加到细胞选择性培养基上无菌落形成。 </li><li>随机挑选了几个数转化到YPD培养基殖民地获得单菌落分离。等待两天菌落生长，然后采取几个单菌落（至少5次），使YPD在选择性培养基上的补丁。 </li><li>设计一对引物扩增区域（250-1,000 BP）的菌落PCR（图1B），含有RNA的寡核苷酸的目标。菌落PCR的步骤如下（Storici和雷斯尼克修改，2006 年 2） 。 <ol><li>从单个修补程序的50μL水悬浮细胞（大约1 mm3的），并添加1个单位的lyticase。在室温下孵育10分钟，随后在一个加热块孵化100 ° C为5分钟，打破了细胞和基因组DNA在溶液中释放。 </li><li> PCR条件：PCR反应包括10μL的细胞再悬浮溶液，正向和反向引物各50 pmoles，1μL10毫米dNTPs，1个单位的Taq DNA聚合酶，5μL10倍的缓冲和调整用无菌水到终体积50μL。 PCR程序是3分，30个循环95章第30 ° C °，55章第30，在72分钟和1℃;的最后72℃延伸7分钟时间° C，然后样品在95℃保存在4 ° C。这种反应的一个延伸时间是1分/ KB假设。 </li><li>继PCR检测，样品的1％或2％（取决于预期大小的PCR产物）观察PCR产物琼脂糖凝胶（图3）上运行。 </li></ol></li><li>如果遗传信息的RNA含有寡核苷酸转移产生的一个新的限制在酵母基因组的目标区域网站（图1B），它是可以用适当的限制性内切酶消化PCR产物，以核实信息的正确传输。如果没有任何限制的网​​站是由RNA含寡核苷酸的生成，请转至步骤6。精华PCR产物使用特定的限制性内切酶。消化反应包括6μLPCR产物，缓冲区，牛血清白蛋白（可能不需要某些酶，所用的酶的指令），限制性内切酶0.5μL，无菌水15μL。样品孵育的温度所用的酶的特定的1小时。 </li><li>运行未消化的样本连同同一行2％琼脂糖凝胶上的消化样品观察遗传修饰的RNA的RNA含寡核苷酸（图3）道转移。 </li><li>使用PCR纯化试剂盒纯化PCR产物和DNA测序作好准备。提交样本用于放大的产品相同的引物序列。 </li><li>分析DNA测序结果，使用软件，它允许多个序列与选择的参考序列（图4）对齐。 </li></ol> E.碱处理RNA含寡核苷酸（图5） <ol><li>对于每一个反应，加入到1.5 ml管nmole 1 RNA含寡核苷酸，或DNA的寡核苷酸（4 250 pmolesμL/μL原液）。 </li><li>加入4μL的1 M氢氧化钠进行水解，或另外添加4μLH 2 O为阴性对照，并在65℃水浴中孵育1小时然后将冰水浴。 </li><li>与2μL1.2 M盐酸，1中号的Tris - HCl，4μLH 2 O 4μL，或H 2 O和另外6μL，4μL1米的Tris - HCl为阴性对照压制。在冰上保持到转型。 </li></ol><img src="/files/ftp_upload/2152/2152fig1.jpg" alt="图1" /> 图1。有缺陷的trp5基因和基因的RNA含有寡纠正TRP5示意图一）trp5突变基因包含2碱基缺失（黑色三角形）和1基义突变（星号）。的单链RNA含插入2基“（蓝色回路）和1 - RNA的碱基替换（红色矩形）生成一个 Van91我限制性内切酶（支架）寡核苷酸酵母细胞转化为正确的遗传缺陷的trp5基因。 b）在TRP5基因是由RNA含寡核苷酸（蓝色矩形表示更正基地）修复，Van91我的网站是到TRP5基因产生的。一个278 bp的片段，包括只有一个Van91我TRP5基因的酶切位点，是PCR扩增，由一对引物（P1和P2）。 P1和P2的Van91 PCR产物消化后产生的177 bp和101 bp的片段，我也所示。 <img src="/files/ftp_upload/2152/2152fig2.jpg" alt="图2" /> 图2。含有的RNA寡核苷酸转化的结果。）无寡核苷酸转化的酵母细胞不形成SC -色氨酸中的任何殖民地，看到板AB 。 B）板CG显示酵母殖民地SC -色氨酸越来越多后，细胞转化与1 RNA含寡nmole。 C）板HL显示酵母菌菌落SC -色氨酸越来越多后，细胞转化与相应的DNA只控制寡核苷酸1 nmole。 <img src="/files/ftp_upload/2152/2152fig3.jpg" alt="图3" /> 图3。检测遗传信息转移到RNA的寡核苷酸经酶切消化的PCR扩增目标基因组区域。P1和P2扩增，并与 Van91我限制性内切酶消化PCR检测样本的2％琼脂糖凝胶电泳产品含有酵母染色体DNA。泳道1，8和15，尺寸为100，200，300，400和500 bp的DNA阶梯上标明左巷，2巷3，PCR扩增PCR 产物 trp5突变株的基因组DNA扩增trp5轨迹产品从激进党+唯一的DNA寡针对性的殖民地的基因组DNA扩增;巷4-7来自色氨酸+含有RNA的寡有针对性的殖民地的基因组DNA; 9日至14巷，Van91我酶切扩增，PCR产物从2至7车道的PCR产物。 Van91我的完整无缺的PCR产品在10至14车道的乐队的存在可以解释TRP5轨迹（CCACATTCTGG）部分消化。考虑到切割现场Van91我（CCANNNN NTGG）可以有多个序列，在TRP5生成的网站可能无法酶的最优化的目标。事实上，所有上述的PCR产物以下的DNA测序，我们没有额外的变化超出寡核苷酸进行（见图4）的检测。 Van91 P1和P2引物和消化产品带扩增278 bp的PCR乐队177 bp和101 bp的我都显示在右边的箭头。 <a href="http://www.jove.com/files/ftp_upload/2152/2152fig4.jpg"><img src="/files/ftp_upload/2152/2152fig4tmb.jpg" border="0" alt="图4Thumb" /></a> 图4。 DNA测序结果显示含有的RNA寡核苷酸基因校正。针对一个基因组区域）的DNA电泳的RNA含寡核苷酸。 RNA含寡核苷酸的RG的DNA序列（蓝色盒装）摹来自。此外盒装的是插入的CG基地。从所有其他的PCR产物的测序结果也为骑士AR为这一个远高于背景的荧光信号，。 B）的的色氨酸+的的DNA，只有寡核苷酸（T1），和的RNA，含寡核苷酸（T2 - T5）的顶部共识序列匹配的针对性和转化 TRP5地区的序列是比较的trp5突变细胞针对寡核苷酸（基因组DNA，前红色阴影部分）。修复RNA含在底部的寡核苷酸DNA插入双基和一个碱基（G）的RNA的替代标记为红色。地区盒装在蓝色与黄色渐变显示的RNA以及含有寡核苷酸的DNA寡核苷酸正是纠正在所有被测样品的缺失突变和无意义突变。虚线标记的RNA寡核苷酸序列的位置。 <img src="/files/ftp_upload/2152/2152fig5.jpg" alt="图5" /> 图5。碱处理可以防止基因的寡核苷酸的RNA含校正，由RNA含寡核苷酸（R）的转化频率是在红色的DNA寡核苷酸（四）所示，在蓝色酒吧的酒吧和所示。误差棒代表3寡核苷酸为每个独立的转换的平均值的标准误差。的DNA寡核苷酸显示没有用NaOH处理类似的转换频率。不同，改造RNA含寡核苷酸的频率下降到0以下用NaOH处理。因此，与RNA含寡核苷酸的准备不是只与DNA寡核苷酸污染，因此，观察到的转换频率是特定的RNA含有寡。

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	<li>Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A., Resnick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-templated+DNA+repair.">RNA-templated DNA repair.</a> Nature. 447, 338-3341 (2007).</li><li>Storici, F., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+delitto+perfetto+approach+to+in+vivo+site-directed+mutagenesis+and+chromosome+rearrangements+with+synthetic+oligonucleotides+in+yeast.">The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.</a> Methods Enzymol. 409, 329-345 (2006).</li><li>Storici, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+DNA+modifications+and+RNA-templated+DNA+repair.">RNA-mediated DNA modifications and RNA-templated DNA repair.</a> Curr Opin Mol Ther. 10, 224-230 (2008).</li><li>Cerritelli, S. M., Crouch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+H:+the+enzymes+in+eukaryotes.">Ribonuclease H: the enzymes in eukaryotes.</a> FEBS J. 276, 1494-1505 (2009).</li></ol>

本文的视频制作赞助商赛默飞世尔科技，产生本出版物中使用的试剂和仪器。

事实上，RNA可以直接传输的遗传信息在细胞基因组DNA被发现利用使用合成的RNA含寡核苷酸（Dharmacon合成的寡核苷酸）1。这是原则的证明，细胞可以作为DNA合成的模板中使用含RNA分子或RNA的唯一序列。使用RNA，含有不仅导致示威从RNA遗传信息可直接传送，而不需要一个反转录DNA复制中间的基因组DNA寡核苷酸，但也认为RNA可以作为同源模板使用证明1,3的DNA损伤的修复。寡核苷酸的RNA只或只包含在DNA序列中嵌入一个单核苷酸，在这个例子中，我们这里介绍。 RNA含寡核苷酸具有一个嵌入式设计，以纠正基因缺陷 trp5等位基因在的无义突变的核苷酸。从核苷酸基因组DNA的遗传信息传递的是显示在表型的改变目标细胞（细胞的缺乏色氨酸的培养基上生长）。只有一个控制的DNA寡核苷酸与RNA含有寡基因校正的频率测量和比较。通过执行此基因在不同的单元格背景，在这里我们酵母基因变异各种校正实验中，我们可以检测因子/ s的具体影响，针对含有RNA的寡核苷酸。因此，我们可以揭示细胞如何调节的RNA / DNA杂交的稳定机制。 ，我们可以通过简单的设计含有RNA的寡核苷酸的不同变种确定一个特定的RNA为模板，在DNA修饰的道的可能性，我们可以判断嵌入到DNA的特定RNA序列的稳定性。此外，我们可以找出是什么在体内 RNA / DNA杂交相互作用的因素基板的首选。 虽然几个在体外研究中，主要是利用短RNA寡核苷酸，已进行了表征因素的功能，能够识别DNA的RNA混合，如核糖核酸 酶H酶4在体内核糖核酸酶H功能，以及其他可能影响的RNA / DNA杂交的稳定仍然大多是未知的蛋白质的身份。利用的可能性RNA包含一个相当长（50至80个碱基）和最优的质量的寡核苷酸（如包含的Thermo Scientific Dharmacon的RNA寡核苷酸）打开的方式直接在体内研究的分子过程的广泛细胞的兴趣。此视频，转换，利用RNA寡核苷酸要求基本上只有一个相比，使用DNA分子的改造，以防止RNase的降解一些额外的步骤。因此，改造使用的RNA寡核苷酸不限酵母系统，但可以适用于任何生物体或细胞类型的DNA寡核苷酸的转型是精通。 总之，针对细胞含有RNA寡核苷酸生成的RNA / DNA杂交和嵌入的DNA在细胞体内的核糖提供了机会。的稳定性，功能和在体内的这些后果产生的RNA / DNA杂交，可分析和特点，潜在的发现未知的DNA修复机制，揭示新的战略目标基因。

答：转换试剂和媒体（从Stor​​ici和雷斯尼克修改，2006年<ol><li> YPD（酵母蛋白胨葡萄糖）：1升，葡萄糖20克，20克大豆蛋白胨10克酵母提取物。添加15克琼脂，使YPD固体培养基。使用高压灭菌器前。在室温下储存。 </li><li>解决方案1：0.1的醋酸锂中号。改造前立即准备。解决方案1是一个可行的解决方案，因此，它是准备直接从粉末。没有股票的解决方案是。保持室温。 LiAc增加酵母细胞壁的通透性，DNA。 </li><li>解决方案2：0.1 M的醋酸锂和聚乙二醇4000的50％。 2解决方案是一个可行的解决方案，它是直接从粉末。没有股票的解决方案是准备。保持室温。 PEG的存款到酵母细胞中的寡核苷酸。 </li><li>含RNA寡核苷酸（的Thermo Scientific Dharmacon），50 80个碱基，脱盐，deprotected和非纯化。重悬，以250 pmoles /μL。储存在-80 ° C。 </li><li>唯一的DNA寡核苷酸，50 80个碱基，脱盐和非纯化。到50 pmoles /μL重悬。储存在-20 ° C。 </li><li> SC -色氨酸（合成完整的媒体缺乏色氨酸）固体介质。 </li><li>直径0.5厘米的玻璃珠，高压灭菌消毒。 </li><li>核糖核酸酶小康：核糖核酸酶净化解决方案。 </li><li> DNA酶/无RNase的，无菌的离心管。 </li><li> DNA酶/无RNase，无菌锥形管。 </li><li> DNA酶/无RNase，无菌气溶胶与ZAP枪头：1-200毫升，100〜1000毫升。 </li></ol> B.菌落PCR材料。 <ol><li> DNA引物，脱盐和非纯化。 50 pmoles /μL无菌水溶解。储存在-20 ° C。 </li><li> Taq DNA聚合酶，缓冲液，dNTPs。 </li><li> PCR管。 </li></ol> C. PCR纯化。 <ol><li> PCR纯化试剂盒。 </li></ol> D.凝胶电泳。 <ol><li>琼脂糖。 </li><li> 1 × TBE缓冲液（45毫米的Tris -硼酸盐和1毫米乙二胺四乙酸）从10倍TBE稀释。 </li><li>预染分子量标记。 </li><li> DNA上样染料。 </li></ol> E.酶切。 <ol><li>限制性内切酶，10X缓冲区，BSA。 </li></ol> F.碱含有的RNA寡核苷酸治疗。 <ol><li> 1 M的氢氧化钠溶液。 </li><li> 1.2 M的盐酸溶液。 </li><li> 1米的Tris - HCl，pH 7.4的解决方案。 </li></ol>

generation of rna/dna hybrids in genomic dna by transformation using rna-containing oligonucleotides

合成短核酸聚合物，寡核苷酸（寡核苷酸），是分子生物学的功能和最广泛的工具。寡核苷酸可以生产包含任何所需的DNA或RNA序列，可以准备，包括各种各样的基地和糖修改。此外，寡核苷酸，可以设计模仿特定的核酸改变，因此，可以作为服务的重要工具，探讨DNA损伤修复机制的影响。我们发现的Thermo Scientific Dharmacon RNA含有之间50和80个核苷酸长度的寡核苷酸可以在体内，职能和染色体RNA / DNA杂交的后果，并嵌入到DNA的核苷酸，特别适合于研究。可以很容易地形成的RNA / DNA杂交，在DNA复制，修复和转录，然而，很少已知的RNA / DNA杂交细胞和这些混合动力车可以在何种程度上影响细胞的遗传完整性的稳定。因此，含有RNA的寡核苷酸，代表一个完美的载体，引入染色体DNA的核苷酸，并产生选择长度和碱基组成的RNA / DNA杂交。在这里，我们现在到的真核细胞模型系统酵母/啤酒酵母/基因组的核苷酸掺入协议。然而，我们的实验室已利用的Thermo Scientific Dharmacon的RNA含寡核苷酸在染色体水平在不同的细胞系统产生的RNA / DNA杂交，从细菌到人类细胞。

Watch this Scientific Journal Video about Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides at JoVE.com

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

这项工作表明如何形成RNA / DNA杂交和染色体水平揭示基因组DNA在酵母细胞中的遗传信息从RNA转移。

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

generation-rnadna-hybrids-genomic-dna-transformation-using-rna

Research

JoVE Journal

Biology

26.7K 次观看.  Georgia Institute of Technology. 这项工作表明如何形成RNA / DNA杂交和染色体水平揭示基因组DNA在酵母细胞中的遗传信息从RNA转移。

视频: Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...