这个项目是支持部分由约翰斯·霍普金斯大学，教务长办公室通过网关科学倡议和QIAGEN公司

简介:一个单一的菌落，应使用适当的孵育过夜培养肉汤接种。甲细菌沉淀，然后处理使用市售的基因组提取试剂盒纯化的基因组DNA。纯化的基因组DNA应该有一个260/280（nm）的比率&gt; 1.8，以确保样品是无蛋白污染。生成焦磷酸测序模板所需的基因组DNA的量是10-20纳克。 A.模板的扩增 （PyroMark PCR手册; <a href="http://www.qiagen.com/handbooks" target="_blank">www.qiagen.com /手册</a> 1） 设置PCR反应 注:一个引物必须在其5&#39;端生物素化，它的建议，是为模板制备高效液相色谱纯化。我们建议PyroMark PCR和SE检测设计软件2.0优化短读测序的序列设计引物，然而，底漆，的BLAST（ <a href="http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome" target="_blank">http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome</a> ）也可以从NCBI用于引物设计。 <ol><li>解冻所有所需的解决方案（ 表1中列出），并混合每一个彻彻底底。根据表1的反应。所有的工作都可以在室温下进行。 </li></ol> 注意:PCR扩增预混试剂含有MgCl 2的终浓度为1.5mM的，在大多数情况下提供令人满意的结果。如果需要更高的Mg 2 +浓度是必需的，可以被添加到高达3.5微升的25mM的MgCl 2反应。 <ol start="2"><li>充分混合反应混合物轻轻吹打向上和向下，然后分配适当的卷PCR管。 </li><li>添加templat"每个PCR管êDNA。我们建议10 ng的基因组DNA。 </li><li>方案的热循环仪，按照表2中。最后一个保持在4℃，建议方便。如果使用热循环仪没有热盖，用100μl矿物油覆盖反应。 </li><li> PCR管放置在PCR仪中，并开始循环程序。 </li></ol> B.真空工作站协议固定的生物素标记的PCR产物<ol><li>根据图4，使主结构。 </li></ol> 注:移液之前，链霉亲和素涂层琼脂糖珠轻轻摇晃瓶子，以确保均匀的悬浮液。 <ol start="2"><li>根据使用的PCR产物的体积，取60-75微升的PCR板的各孔中，以使每孔的总体积为80微升混合液到。 </li><li>向每孔中加入5-20μl的生物素化的PCR产物。 </li><li>密封条带长约井ps和PCR板在1400 rpm搅拌5-10分钟，在室温下（15-25℃），使用轨道摇床。 </li></ol>变性的DNA和测序引物的添加<ol><li>用退火缓冲液稀释的测序引物，以0.3μM和分配到一个反应板的各孔中25微升。定位板在工作站上。 </li><li>根据图5的图，填充工作站槽。 </li><li>开启真空泵，确保真空工具的开关设置（ 图6）。冲洗过滤器探针槽5的高纯度水（Milli-Q系统18.2兆瓦为x cm或同等学历）（ 图5）。在步骤12中使用新鲜的高纯度水笔芯的低谷。 </li><li>工作迅速，定位的PCR板珠在工作站上。确保两板在同一方向时，将样品加载。 </li><li>随着真空开关ON，降低真空工具的PCR板入井，持续20秒或直到所有音量都已经被吸。通过真空工具（ 图7），将被捕获的有孔玻璃珠。 </li><li>随着真空，冲洗工具，用70％乙醇（槽1），持续20秒或直到所有的音量已经吸气。 </li><li>真空开，冲洗工具变性溶液（槽2），持续20秒或直到所有卷已经被吸。 </li></ol> 注意:该变性溶液含有氢氧化钠，这是眼睛和皮肤的刺激性。总是穿白大褂，手套和护目镜。 <ol start="8"><li>真空开，用洗涤缓冲液（槽3）冲洗工具，持续20秒或直到所有音量都已经被吸。 </li><li>真空开，真空工具提高到超过90°垂直，持续5秒，让所有的液体排出真空工具。关闭循环泵停止，并对齐与反应板的真空工具。降低进入真空工具井，轻轻摇晃从一边到另一边释放珠入井含有测序引物。 </li><li>为了清洁真空工具，搅拌过滤器探头在高纯度的水（4槽），持续10秒。 </li><li>真空切换和刷新工具用高纯度的水（5槽），持续20秒或直到所有音量都已经被吸。 </li><li>真空工具提高到超过90°C垂直范围为5秒，然后真空开关关闭，并存储在"停车"位置。 </li></ol>测序引物退火到DNA链<ol><li>在预热的板固定器，将反应板。 </li><li>加热板在80℃下加热块上，持续2分钟。 </li><li>拆下该板从固定器在柜台上，在室温下冷却5分钟。 </li></ol> Pyromark Q24操作 注:以上的电源线使用电源开关位于仪器上的开关。明星锤头可能需要长达1分钟。 <ol><li>填充墨盒PyroMark黄金Q24试剂手册中提供的指示。要确定需要，设置了仪器软件运行文件的卷，并在"工具"菜单下，选择预运行信息</li><li>如果仪器已经开启，确保它不执行运行，然后打开盖子。将发出可听见的警告信号，如果盖子被打开时，这样做是不安全的。 </li><li>打开墨盒门和插入墨盒标签朝前（ 图8）。确保墨盒已正确插入（一条线在前面应该是可见的），并关闭大门。 </li><li>打开板保持框和定位仪器内部的样品板。 </li><li>关闭平板保持框和仪器的盖子（ 图9）。 </li></ol>启动运行<ol><li>插入USB记忆棒运行文件Ş仪器软件创建到USB端口在前面的仪表。 </li><li>在主菜单中选择"运行"，然后按"确定"。 </li><li>使用滚动箭头来选择所需的运行文件，按"选择"。 </li></ol> 注:该仪器将开始分配时达到所有预设的压力和温度水平（这可能需要几分钟）的试剂。在运行过程中，仪​​器屏幕上显示的实时选择的热解图。使用滚动箭头来查看其他井的热解图。 完成一个运行<ol><li>当仪器确认，程序运行结束并运行文件已被保存到U盘，按"关闭"。 </li><li>取下USB记忆棒。如果它被删除完成运行前，插入USB记忆棒。选择"管理"，然后"复制未保存的运行"。 </li><li>现在运行的文件可以被打开和使用仪器软件分析和比较的库已知微生物使用Identifire软件的。 </li></ol>

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	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PyroMark PCR Handbook. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PyroMark Q24 User Manual. , (2012).</li><li>Elahi, E., Ronaghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+a+tool+for+DNA+sequencing+analysis.">Pyrosequencing: a tool for DNA sequencing analysis.</a> Methods Mol. Bio. 255, 211-219 (2004).</li><li>Fakhrai-Rad, H., Pourmand, N., Ronaghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+and+accurate+detection+platform+for+single+nucleotide+polymorphisms.">Pyrosequencing: and accurate detection platform for single nucleotide polymorphisms.</a> Hum. Mutat. 19 (5), 479-485 (2002).</li><li>Langaee, T., Ronaghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+analyses+by+pyrosequencing.">Genetic variation analyses by pyrosequencing.</a> Mutat. Res. 573 (1-2), 96-102 (2005).</li><li>Liu, Z., Lozupone, C., Hamady, M., Bushman, F. D., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+pyrosequencing+reads+suffice+for+accurate+microbial+community+analysis.">Short pyrosequencing reads suffice for accurate microbial community analysis.</a> Nucleic Acids Res. 35 (18), e120 (2007).</li><li>Novais, R. C., Thorstenson, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+Pyrosequencing+for+microbiology:+From+genes+to+genomes.">The evolution of Pyrosequencing for microbiology: From genes to genomes.</a> J. Microbiol. Methods. 86 (1), 1-7 (2011).</li><li>J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+pyrosequencing+and+microbial+identification.">Metagenomic pyrosequencing and microbial identification.</a> Clin. Chem. 55 (5), 856-866 (2009).</li><li>Quince, C., Lanzen, A., Curtis, T. P., Davenport, R. J., Hall, N., Head, I. M., Read, L. F., Sloan, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+determination+of+microbial+diversity+from+454+pyrosequencing+data.">Accurate determination of microbial diversity from 454 pyrosequencing data.</a> Nat. Methods. (9), 639-6341 (2009).</li><li>Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M., Nyren, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+DNA+sequencing+using+detection+of+pyrophosphate+release.">Real-time DNA sequencing using detection of pyrophosphate release.</a> Anal. Biochem. 242, 84-89 (1996).</li><li>King, C., Scott-Horton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+A+simple+method+for+accurate+genotyping.">Pyrosequencing: A simple method for accurate genotyping.</a> J. Vis. Exp. (11), e630 (2008).</li><li>Rothberg, J. W., Heinz, , 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=An+integrated+semi-conductor+device+that+enabling+non-optical+genome+sequencing.">An integrated semi-conductor device that enabling non-optical genome sequencing.</a> Nature. 475, 348-352 (2011).</li><li>Levene, M. J., Korlach, J., Turner, S. W., Foquet, M., Craighead, H. G., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zero-Mode+Waveguides+for+Single-Molecule+Analysis+at+high+concentrations.">Zero-Mode Waveguides for Single-Molecule Analysis at high concentrations.</a> Science. 299, 682-686 (2003).</li><li>Eid, J., Fehr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+DNA+Sequencing+from+Single+Polymerase+Molecules.">Real-Time DNA Sequencing from Single Polymerase Molecules.</a> Science. 323, 133-138 (2009).</li></ol>

生产和自由地进入本文由Qiagen公司赞助。 作者艾哈迈德·迪罗谢，杰森和瓦迪Qiagen公司产生在这篇文章中所使用的试剂和仪器的公司员工。

几个新的下一代测序方法已经商品化。三，最广泛使用的方法是离子半导体测序，单分子实时测序，并通过合成测序（SBS）。3-11每一种方法都依赖于通过合成测序，但结合的核苷酸的检测，采用新颖的平台。在离子半导体测序，测序链作为模板的DNA单链供应12聚合酶和核苷酸依次加入焦磷酸测序。当一个核苷酸加入到生长的DNA链中，氢离子被释放。的场效应晶体管基于传感器检测到的氢离子。 单分子实时测序取决于零模式波导（中贸网）13中贸网是一个小的结构，在一个单一的的聚合酶分子被连接到井底。在这样一种方式，一个花神照明气味分子可以被检测到。每个核苷酸用荧光分子标记。作为荧光标记核苷酸注册成立，探测器识别的核苷酸。当添加的下一个核苷酸，荧光分子被裂解14 通过合成测序（SBS）使用一种独特的方法扩增靶DNA等独特的序列的簇生成15要生成并合成互补链的单链模板。每个核苷酸用荧光分子标记和碱加成后，每个记录的添加的碱的荧光。

焦磷酸测序法是一种快速，准确的方法，以"合成测序"的原则是根据序列核酸。 "通过合成的序列"涉及到使用一个单链DNA模板合成互补链的一个基站在一个时间，和检测法团的碱（A，T，G或C）在每个步骤通过检测的化学发光信号。焦磷酸测序反应包括模板DNA的酶，dNTPs（三磷酸的dATP，dTTP的，dCTP标记），DNA聚合酶，ATP硫酸化酶，萤光素，萤光素酶，三磷酸腺苷双磷酸酶。过程是由一个另外的四种dNTP和DNA聚合酶的生物素化的单链模板DNA的合成反应。 DNA聚合酶结合到模板上的互补的dNTP，与随后释放的焦磷酸盐（ 图1）。 ATP硫酸按比例转换成的焦ATP。 ATP作为氧化萤光素，荧光素的荧光素酶介导的转化用催化剂产生光（ 图2）。掺入 ​​的核苷酸的数量成比例的光的强度，确定是否一个或多个特定的dNTP（的dATP，dTTP的，三 ​​磷酸，或dCTP标记）是模板链顺序（ 图3）上存在的。任何非法人的dNTP降解三磷酸腺苷双磷酸酶之前，加入下一个dNTP的合成反应的延续。这种"合成测序"反应与另外四种dNTP各重复，直到确定的DNA序列的单链DNA模板。 要查看焦磷酸测序反应的动画， <a href="http://www.jove.com/files/ftp_upload/50405/50405movie1.mov" target="_blank">点击这里观看电影</a> 。

<table border=1>
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>PyroMark PCR Master Mix, 2x</td>
 <td>Qiagen</td>
 <td>978703</td>
 <td></td>
 </tr>
 <tr>
 <td>CoralLoad Concentrate, 10x</td>
 <td>Qiagen</td>
 <td>978703, 978705</td>
 <td></td>
 </tr>
 <tr>
 <td>Q-Solution, 5x</td>
 <td>Qiagen</td>
 <td>978703, 978705</td>
 <td></td>
 </tr>
 <tr>
 <td>MgCl2, 25 mM</td>
 <td>Qiagen</td>
 <td>978703, 978705</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase-Free Water</td>
 <td>Qiagen</td>
 <td>129112</td>
 <td></td>
 </tr>
 <tr>
 <td>Primers</td>
 <td>Qiagen</td>
 <td>978776 or 978777</td>
 <td>Primers should be purchased from an established oligonucleotide manufacturer (i.e. QIAGEN Pyromark Custom Assays, IDT, etc). Lyophilized primers should be dissolved in TE to provide a stock solution of 100 &mu;M.</td>
 </tr>
 <tr>
 <td>Pyromark Gold Q24 Reagents</td>
 <td>Qiagen</td>
 <td>970802</td>
 <td></td>
 </tr>
 <tr>
 <td>High-purity water (Milli-Q 18.2 M&Omega; x cm or equivalent)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Streptavidin Sepharose High Performance Beads</td>
 <td>GE Healthcare</td>
 <td>17-5113-07</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Annealing Buffer</td>
 <td>Qiagen</td>
 <td>979009</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Denaturation Solution</td>
 <td>Qiagen</td>
 <td>979007</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Wash Buffer</td>
 <td>Qiagen</td>
 <td>979008</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24</td>
 <td>Qiagen</td>
 <td>9001514</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24 Cartridge</td>
 <td>Qiagen</td>
 <td>979202</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q96 HS Tip Holder Box</td>
 <td>Qiagen</td>
 <td>979105</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24 Vacuum Workstation</td>
 <td>Qiagen</td>
 <td>9001516</td>
 <td></td>
 </tr>
 <tr>
 <td>PyroMark Q24 Plate Holder</td>
 <td>Qiagen</td>
 <td>9022273</td>
 <td></td>
 </tr>
 <tr>
 <td>Orbital shaker</td>
 <td>Fisher</td>
 <td>11-675-297</td>
 <td></td>
 </tr>
 <tr>
 <td>Heat block</td>
 <td>Fisher</td>
 <td>11-718Q</td>
 <td></td>
 </tr>
 </tbody>
</table>

pyrosequencing for microbial identification and characterization

焦磷酸测序法是一种通用的技术，可用于确定细菌种类，歧视细菌菌株，并检测抗微生物药物抗性的基因突变，有利于微生物的基因组测序。焦磷酸测序微生物应用程序的优点包括快速和可靠的微生物和微生物基因组突变的高通量筛选和准确的鉴定。焦磷酸测序法测序的DNA合成的互补链在一个时间一个单独的基，而确定的特定核苷酸的合成反应过程中被纳入。该反应发生在固定的单链模板DNA的四种脱氧核糖核苷酸（dNTP的）中依次加入下一个dNTP的合成反应，然后加入酶降解，并非法团的dNTPs。被由一代chemilum监测检测模板纳入特定的碱基信号inescent。的dNTPs，产生化学发光信号的顺序决定的模板的DNA序列。焦磷酸测序技术使实时测序能力快速微生物鉴定，在一个单一的检测。此外，焦磷酸测序仪，分析抗微生物药物耐药性的，包括分型的SNP位点，点突变，插入，缺失，以及定量分析中可能发生的一些抗微生物性的多基因拷贝的完整遗传多样性图案。

使用该软件的一个典型的焦磷酸测序运行的结果显示的位置的正向和反向引物，用于扩增子的产生，和保守的16S核糖体序列（ 图10）内的焦磷酸测序反应所用的测序引物，引物之间的序列的高变网站允许了大量的细菌种类，使用保守的测序引物（5&#39;-TACATGCAAGTCGA）进行细菌鉴定。作为一个内部的质量控制中，可变区的DNA序列包围可以被检查，以确保了正确的DNA区域已被分析。焦磷酸测序软件允许到内部数据库中进行细菌鉴定细菌核糖体序列生成的序列的比较和调整。此外，序列可以被分析以确定赋予抗生素耐药性的突变。例如，突变分析的23S核糖体基因Helicob字符幽门螺杆菌演示了两种抗生素抗性的突变模式（GAA或AGA）（ 图11）。分析多株来自同一病人可同时检测跟踪耐药性的出现，随着时间的推移，以帮助微生物耐药性爆发的流行病学调查。 <img alt="图1" src="/files/ftp_upload/50405/50405fig1.jpg" /> 图1。使用生物素化的PCR产物作为模板，将导致生成的焦磷酸（PPi）的DNA聚合酶，dNTPs浓度。 <img alt="图2" src="/files/ftp_upload/50405/50405fig2.jpg" /> 图2。 ATP硫酸按比例转换成焦磷酸，三磷酸腺苷，三磷酸腺苷的催化剂作为荧光素的荧光素酶介导的转化率氧化萤光素，产生光，是亲正比于ATP的量。记录光热解图跟踪峰值，并表示核苷酸成立。非法人的dNTPs先下一加入的dNTP继续合成三磷酸腺苷双磷酸酶降解。 <img alt="图3" src="/files/ftp_upload/50405/50405fig3.jpg" /> 图3。如果一个或多个特定的dNTP（的dATP，dTTP的，三 ​​磷酸，或dCTP标记）被合并到模板链上的顺序所产生的光的强度。 <img alt="图4" src="/files/ftp_upload/50405/50405fig4.jpg" /> 图4。预混固定生物素化的PCR产物编制流程图。 <img alt="图5" src="/files/ftp_upload/50405/50405fig5.jpg" /> GE ="总是"&gt; 图5。 PyroMark工作站，PyroMark板，PCR板，和波谷的位置。 <img alt="图6" src="/files/ftp_upload/50405/50405fig6.jpg" /> 图6。真空开关ON和OFF位置的位置。 <img alt="图7" src="/files/ftp_upload/50405/50405fig7.jpg" /> 图7。真空工具。正确处理真空工具。 <img alt="图8" src="/files/ftp_upload/50405/50405fig8.jpg" /> 图8。门打开墨盒与墨盒到位。 <img alt="图9" src="/files/ftp_upload/50405/50405fig9.jpg" /> 图9所示。正确插入墨盒门关闭。 "FO:保持together.within"="总是"&gt; <img alt="图10" src="/files/ftp_upload/50405/50405fig10.jpg" /> 图10。基于焦磷酸测序的细菌鉴定结果，设计PCR引物保守区域的DNA模板和测序引物的位置的紧上游，其特征在于良好的吞吐量高变的DNA序列内的扩增子（显示为蓝色）。 <img alt="图11" src="/files/ftp_upload/50405/50405fig11.jpg"/> 图11。使用焦磷酸测序幽门螺杆菌抗生素耐药检测 23S含有GAA AGA序列的幽门螺杆菌基因，赋予抗菌性的突变分析。 <a href="https://www.jove.com/files/ftp_upload/50405/50405fig11large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1"><tbody><tr> &lt;TD&gt; 组件 </td><td> 每个反应体积 </td><td> 终浓度 </td></tr><tr><td>火焰兵PCR预混，2 </td><td> 12.5微升</td><td> 1X </td></tr><tr><td>的浓缩CoralLoad，10倍</td><td> 2.5微升</td><td> 1X </td></tr><tr><td> 25毫米氯化镁2（可选） </td><td>变量</td><td> ≥1.5毫</td></tr><tr><td> Q-解决方案，5（可选） </td><td> 5微升</td><td> 1X </td></tr><tr><td>引物A /底漆B </td><td>变量/变量</td><td> 0.2μM/0.2微米</td></tr><tr><td> RNase-free的水</td><td>变量</td><td> - </td></tr><tr><td> 总成交量（后加入模板DNA） </td><td> 25微升 </td><td></td></tr></tbody></table> 表1中。 PCR反应混合物。 <table border="1"><tbody><tr><td></td><td> ＆NBSP; </td><td></td><td> 补充意见 </td></tr><tr><td>最初的PCR活化步骤</td><td> 15分钟</td><td> 95°C </td><td> HotStartTaq DNA聚合酶被激活</td></tr><tr><td> 3步法循环:变性</td><td> 30秒</td><td> 94°C </td><td></td></tr><tr><td>退火</td><td> 30秒</td><td> 60°C 56°C </td><td>对于基因组DNA 亚硫酸氢盐转化的DNA </td></tr><tr><td>延期</td><td> 30秒</td><td> 72°C </td><td></td></tr><tr><td> 的周期数 </td><td> 45 </td><td></td><td></td></tr><tr><td> 最后延伸 </td><td> 10分钟</td><td> 72°C </td><td></td></tr></tbody></table> 表2中。 PCR循环规范。

Watch this Scientific Journal Video about Pyrosequencing for Microbial Identification and Characterization at JoVE.com

Pyrosequencing for Microbial Identification and Characterization

焦磷酸测序法是一种通用的技术，可用于确定细菌种类，区分细菌的菌株，并检测抗微生物药物抗性的基因突变，有利于微生物的基因组测序。在这段视频中，微生物扩增子，扩增子的焦磷酸测序，DNA序列分析的程序将被证明。

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

pyrosequencing-for-microbial-identification-and-characterization

Research

JoVE Journal

Biology

47.0K 次观看.  Johns Hopkins University. 焦磷酸测序法是一种通用的技术，可用于确定细菌种类，区分细菌的菌株，并检测抗微生物药物抗性的基因突变，有利于微生物的基因组测序。在这段视频中，微生物扩增子，扩增子的焦磷酸测序，DNA序列分析的程序将被证明。

视频: Pyrosequencing for Microbial Identification and Characterization

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

科研

生物学

Biology of Microbial Communities  - Interview

微生物群落的生物 - 面试

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...