我们感谢协助取得标本艾伦·塔斯克（美国农业部动植物检疫局，河谷，MD），斯蒂芬·康普顿（克莱姆森），雪利酒Aultman（克莱姆森），克雷格·拉姆齐（美国农业部动植物检疫局，柯林斯堡，CO），和贝蒂Marose（UMD格式） 。我们感谢他的工作在拍摄视频的学生安德鲁·阿德里安（UA-亨茨维尔）和德里克·萨克尔（UA-亨茨维尔）为协助他们在测试此协议，和Herdy约瑟夫。这项工作是由国家鱼类和野生动物基金会。

1。标本采集和保存这种方法是使用新鲜的，冷冻，最近干叶组织开发和测试。 <ol><li>确定茅草和/或JBG草种鉴定专业的分类学家的帮助下组织。其鲜艳的红叶，观赏JBG很容易直观地从野生型的茅草和JBG区分恢复;然而，茅草和JBG恢复从一个几乎没有区别。茅草和恢复的JBG型有绿色，叶片较长，相当大的和较长的根茎，叶面积超过12 JBG（ 图1）。 </li><li>鲜叶组织提供最丰富的和最高质量的DNA，可以从田间或温室种植一收集白茅植物。如果是用于新鲜组织，收集3小时内提取DNA，以帮助防止退化。否则，准备仓库，keepi及组织吴组织的凉爽和阳光直射。 </li><li>存放在日后组织DNA提取，最理想的方法是冻结并保存在-80组织°C间立即。不要让冷冻组织解冻之前提取DNA。转移到DNA提取研磨步骤，以防止解冻之前液氮冷冻组织。 </li><li>如果在-80°C冰箱无法使用，立即干组织。对于干式贮存，一纸信封放到组织和脱水的硅胶或其他活性干燥剂在室温中存放的信封。混有少量指标二氧化硅与非指示硅胶确保二氧化硅是完全脱水，适合干燥植物组织。 </li><li>使用至少10倍以上的重量鲜叶组织硅胶。植物组织要干24小时内， 随着时间的推移，通过干式贮存的DNA的质量和数量的减少（植物标本馆标本的情况下S）。 </li></ol> 2。 DNA的提取从植物组织中提取DNA，按照的DNeasy植物Mini试剂盒（QIAGEN，瓦伦西亚，加州;猫＃69104或69106）制造商的指示，有一个轻微的修改。使用每列建议小于100毫克新鲜组织或低于20毫克干组织，而不是磨大于100毫克，然后转移到新鲜或冷冻组织100毫克（或&gt; 20毫克干组织）适当的管提取。同时核和叶绿体DNA提取。 <ol><li>启动这些程序之前，验证，乙醇添加到AP3的缓冲区/ E和AW。 </li><li>磨细粉，用在冷藏杵臼研磨三轮液氮&gt;新 ​​鲜或冷冻的叶组织（或干叶组织20毫克）100毫克。 中断的起始原料不足或不足裂解也可以导致DNA的产量较低。仔细研磨的TISSUE和不超载太多组织列。 </li><li>转让从新鲜或冷冻组织（或粉干组织20毫克），冻粉100毫克至1.5 ml离心管，400μLAP1的缓冲和4μl的RNase A.的每一管可以放置在一个小机架上平衡来监测每管组织的正确重量 。 </li><li>涡或震动样品（S）混合并孵育10分钟，在65°C时，反相孵化期间的2-3倍管（S）。 </li><li>每个样品中加入130μL缓冲AP2的。混合反相管（S）数次，并在冰上孵育5分钟。 </li><li>吸取各裂解成一个单独的QIAshredder小型离心柱，并将其放置在一个2 ml收集管（试剂盒提供）的每一列。离心柱（S）为20,000 XG（〜14,000 RPM）2分钟，每一个新的管（不与试剂盒提供）流过分数将不破坏任何形成的颗粒转移。 </li><li>加入1.5体积的AP3的缓冲区/E和混合移液。 </li><li>转移到DNeasy小型离心柱在2 ml收集管650μL混合物。在6000 XG（8000转）1分钟，并丢弃流过离心柱（S）。重复此步骤，每个样品的剩余混合物。 </li><li>放入一个新的2 ml收集管（S）的自旋列（S），每一列的顶部添加μl缓冲胡的500。在6000 XG（8000转）1分钟，并丢弃流过离心柱（S）。 </li><li>加入500μl到另一个缓冲区胡每一列的顶部。 20,000 XG（〜14,000 RPM）离心2分钟。这一步将干之列，从而消除任何缓冲区，可以抑制PCR技术中的残留的乙醇。 </li><li>每个离心柱转移到一个新的1.5 ml离心样品管。加入100μL缓冲AE洗脱每一列的顶部，孵化列（S）在室温下5分钟。离心柱（S）在6000 XG（8000转）收集的DNA为1分钟。 </li><li>重复这些洗脱步骤一次，到相同的1.5 ml离心管产量200μL样品洗脱的DNA。存储在-20℃直至使用的DNA样本，DNA的浓度取决于组织的类型和储存条件。获得最佳产量时洗脱了200μl缓冲AE的总DNA，但浓度可以增加洗脱体积减少到低至50μL。 </li></ol> 3。 DNA的质量和数量的核查<ol><li>测试之前PCR设置使用分光光度计或荧光和凝胶电泳提取DNA的质量和数量。这将有助于确保成功的后续步骤。 </li><li>使用分光光度计，检测DNA的质量和数量。良好的DNA产量应该在50和150之间，260/280和230/280比值接近2.0纳克/微升。作为一个例子， 图2显示了良好的质量结果，使用的NanoDrop分光光度计（ThermoScientific<html>威尔明顿，DE）。 </li><li>使用标准的1％琼脂糖凝胶进行电泳。确认没有RNA污染小的裸奔（ 图3）存在比较大的波段（10 KB）。 </li><li>如果DNA浓度高，稀的DNA样本，以70毫微克/微升后续步骤。 </li></ol> 4。 PCR引物在本协议所使用的PCR引物的基础上茅草和JBG基因型之间的叶绿体trnL - F间隔区序列差异。这些差异在形式的SNPs（单核苷酸多态性）而INDELs（插入和删除），允许发展的各种特异引物，通过独特的序列（ 图4）的网站定位的引物。 <ol><li>引物的质量，可以对PCR结果产生重大影响。为了引物从一个有信誉的公司。我们斜生物，公司订购的引物（:/ / www.obliquebio.com/web/“的目标=”_blank“&gt; http://www.obliquebio.com/web/，汉茨维尔，AL），要求脱盐过程的唯一标准。 </li><li> trnL - F阳性对照引物：的底漆集放大的叶绿体trnL - F间隔区最基层类群11-13作为一个很好的阳性对照（产生一个890 bp的条带）。 <table border="1"><tbody><tr><td> 名称 </td><td> 序列 </td></tr><tr><td> trnF基因（GAA） - F </td><td> 5&#39;-ATTTGAACTGGTGACACGAG-3&#39; </td></tr><tr><td> trnL（5&#39;外显子） - C </td><td> 5&#39;-CGAAATCGGTAGACGCTACG-3&#39; </td></tr></tbody></table></li><li> 野生型的茅草引物：这个引物是特定的茅草，不健全JBG基因型trnL-F区。一套带为595 bp的结果。 <table border="1"><tbody><tr><td> 名称 </td><td> 序号uence </td></tr><tr><td> trnLF-C F1 </td><td> 5&#39;-TCCACTTTTTTGAAAAAACAAGTGCAA-3&#39; </td></tr><tr><td> trnLF-C - R1 </td><td> 5&#39;-GCCGATACTCTAATAAATAAAAAAAAAAAAGAAAT-3&#39; </td></tr></tbody></table></li><li> JBG和JBG恢复引子：这个引物是特定JBG基因型和不健全茅草trnL-F区。一套带为594 bp的结果。 <table border="1"><tbody><tr><td> 名称 </td><td> 序列 </td></tr><tr><td> trnLF R-F2 </td><td> 5&#39;-CCAAATCCACTTTTTTGAAAAAACAAGTGGTT-3&#39; </td></tr><tr><td> trnLF-R - R2 </td><td> 5&#39;-CGAGATTCCTTGCCGATACTCTAATAAAA-3&#39; </td></tr></tbody></table></li><li>悬浮在无核酸DDH 2 O的足够量的每个引物获得100毫米的股票，可以储存在-20°C，长期的解决方案。 </li><li>每个引物的股票稀释到12毫米，前PCR设置步骤。 </li></ol> 5。 PCR设置 DNA提取使用上述引物PCR反应扩增。包括阳性对照，以确保所有的PCR试剂运作良好，并可以生成一个乐队。包括阴性对照，以确保没有任何试剂与无用的DNA污染。 阴性对照不包含模板，并应导致没有带生产。 <ol><li>准备薄壁PCR管中的所有反应，以便更好地之间的热循环块和样品的传热。 我们建议使用市售气雾剂无枪头，以帮助避免污染。 </li><li>对于每一个孤立的DNA样本，建立了50μLPCR反应，用0.2毫升薄壁PCR管冰在下面列出的顺序加入下列试剂的每一个以上的引物。如果多个样品正在准备，使该异常包含所有试剂鸡尾酒DNA为模板，建立统一的条件之间的所有反应。 <table border="1"><tbody><tr><td> PCR试 ​​剂 </td><td> 卷使用 </td><td> 最后Concetration </td></tr><tr><td>无核酸DDH 2 O的 </td><td> 40.5微升</td><td></td></tr><tr><td>的10X优势2 PCR缓冲液（Clonetech，CA） </td><td> 5.0μL </td><td> 10％（V / V） </td></tr><tr><td>优势超纯PCR dNTP混合液（10毫米，Clonetech，CA） </td><td> 1.0μL </td><td> 0.2毫米</td></tr><tr><td>引物1（DDH 2 O的 12μm的） </td><td> 1.0μL </td><td> 0.24μM的</td></tr><tr><td> 2底漆（12μm的在DDH 2 O的 ） </td><td> 1.0μL </td><td> 0.24μM的</td></tr><tr><td>优势2聚合酶混合物（Clonetech，CA） </td><td> 0.5μL </td><td> 1％（V / V） </td></tr><tr><td> DNA提取（70毫微克/反应;需要调整DDH 2 O容量） </td><td> 1.0μL </td><td> 1.4纳克/微升</td></tr><tr><td></td><td>总计：50.0微升</td><td></td></tr></tbody></table></li><li>为了确保所有的PCR试 ​​剂运作良好，设置阳性对照，阳性对照引物。 此引物为白茅，JBG同样适用，JBG恢复和其他草，将导致带为890 bp的。 </li><li>设置阴性对照，使用相同的控制底漆作为阳性对照，使用DDH 2 O的 ，而不是DNA的提取。 如果所有试剂的DNA污染，这种反应会导致在没有带。 </li><li>如果DNA浓度低，更多的DNA可以被添加到每个反应，调整无核酸DDH 2 O的使用量，以使反应总体积50μL。 不要使用超过5μL的DNA（10％总体积）每ŕ的反应的影响，尽可能的DNA样本中的杂质会抑制PCR反应。 </li></ol> 6。 PCR循环<ol><li>开展了激烈的盖子使用以下的PCR循环参数配备热循环的PCR扩增。我们使用的Mastercycle亲小号热循环仪（Eppendorf公司，Hauppauge的，纽约州）的设置与标准温度斜坡条件。应执行任何质量热循环。 <table border="1"><tbody><tr><td> 周期 </td><td> 变性退火 </td><td> 聚合 </td></tr><tr><td> 1 </td><td> 2分钟在95℃ </td><td></td></tr><tr><td> 2 </td><td>在95°C 30秒</td><td> 30秒，61°C 90秒68°C间，35个循环</td></tr><tr><td> 3 </td><td></td><td> 5分钟在68°C间</td></tr><tr><td colspan="3">保持在4°C，直到样品被删除</TD&gt; </tr></tbody></table></li><li>优化PCR（包括引物退火温度，延伸时间和循环次数）的条件下，需要的DNA的质量取决于引物，Taq酶或热循环型使用。 我们建议使用渐变功能的热循环时，确定最佳退火温度。 </li><li>如果正在使用的热循环，没有了激烈的盖子，加1滴矿物油，每个样品的顶部，以防止在PCR循环蒸发。 </li></ol> 7。 PCR产物的凝胶电泳可视化的分析结果，单独的1％琼脂糖凝胶使用标准电泳PCR产品。 <ol><li>结合5μL每个PCR扩增产品标准的DNA上样缓冲液2μL（通常是5倍或6倍的解决方案）。 </li><li>加载到1％琼脂糖含ETBR（DNA染色溴化乙锭）凝胶与电子样本ither TAE或SB（四硼酸钠）缓冲系统16。我们使用10毫克/毫升ETBR每100毫升1％琼脂糖（0.1微克/毫升）原液1μL。 </li><li>运行样品〜120V，直到染料前端达到¾凝胶的总长度。 </li><li>在紫外光下（例如短波紫外线灯箱），检查结果乐队，看看是否合适的片段扩增。 </li><li>使用可用的照片，文件系统或相机记录凝胶和由此产生的带。 </li></ol> 8。代表结果可视化的PCR产物后，茅草有JBG或恢复JBG（ 图5）相比，独特的带型。对于每一个DNA样本的trnL - F阳性对照引物应导致在一个单一的高强度〜890 bp的带。这验证所有的PCR试剂运作良好。同样，阴性对照组（无模板）反应应包含不带任何底漆小号等使用。这验证试剂没有受到污染。 如果DNA样本来自野生型的茅草，PCR反应中使用的茅草特异引物集将在约595 bp的单波段的结果，而JBG特异引物将导致在没有带。同样，如果DNA样本是来自的JBG或者恢复JBG，PCR反应中使用JBG特异引物集将导致在〜594 bp的单波段，而茅草特异引物，将导致在没有带。因为JBG和恢复JBG具有相同的核酸序列，因此，他们将有相同的带型。如果有许多样品的凝胶相比，在相同的时间，我们建议运行设置每个引到另一个派生的所有样品，从而使其更容易扫描阳性结果的样品。 JBG和JBG恢复之间的形态差异是相当明显的（如红色的叶子和小身材的JB<html>的Ğ与绿色的色彩，较大的身材和积极成长的JBG恢复），而PCR结果将是相同的，JBG品种很容易区分植物形态。 <img alt="图1" src="/files/ftp_upload/3265/3265fig1.jpg" /> 图1比较，温室种植白茅变种。koenigii（日本血草），恢复了一白茅VAR。koenigii（JBG恢复）和一白茅 （茅草野生型）。 <img alt="图2" src="/files/ftp_upload/3265/3265fig2.jpg" /> 图2。使用的NanoDrop分光光度计验证DNA样本的例子。请注意，不论使用的分光光度计，260/280的比例应该接近1.8 260/230的比例应该接近2.0。 内容】“&gt; <img alt="图3" src="/files/ftp_upload/3265/3265fig3.jpg" /> 图3。DNA样本验证使用标准1％琼脂糖凝胶电泳。大小分析用于商业DNA标记。巷＃4是一个质量较差的DNA样本的例子，显示出涂抹一些RNA污染。 <a href="/files/ftp_upload/3265/3265fig4_lg.jpg"><img alt="图4" src="/files/ftp_upload/3265/3265fig4.jpg" /></a> 白茅变种。koenigii（日本血草），trnL - F区序列图4。恢复一白茅VAR。koenigii（JBG恢复）和一白茅 （茅草野生型）。垂直的黑色箭头表示从单​​核苷酸多态性和个InDel产生的序列的差异。水平的绿色箭头表示野生型的茅草用于茅草-特异PCR引物的位置。水平的红色箭头表示宝JBG和JBG sitions恢复JBG-特异性PCR引物。 <img alt="图5" src="/files/ftp_upload/3265/3265fig5.jpg" /> 图5。代表的JBG与JBG恢复茅草和JBG特异引物的trnL - F阳性对照和无模板的阴性对照以及结合的DNA样本，从茅草所得的PCR产物凝胶电泳结果。

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H., van Santen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+Dynamics+and+Genotypic+Diversity+of+Cogongrass+(Imperata+cylindrica)+at+the+Point+of+Introduction+in+the+Southeastern+United+States.">Invasion Dynamics and Genotypic Diversity of Cogongrass (Imperata cylindrica) at the Point of Introduction in the Southeastern United States.</a> Invasive Plant Science and Management. 1 (2), 133-141 (2008).</li><li>Talley, S. M., Ramsey, C. L., Zink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimentally+assessing+the+invasive+potential+of+plants.">Experimentally assessing the invasive potential of plants.</a> CPHST Laboratory 2009 Annual Report. , 29-30 (2009).</li><li>Hodkinson, T. R., Chase, M. W., Lled&#243;, M. D., Salamin, N., Renvoize, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetics+of+Miscanthus,+Saccharum+and+related+genera+(Saccharinae,+Andropogoneae,+Poaceae)+based+on+DNA+sequences+from+ITS+nuclear+ribosomal+DNA+and+plastid+trnLintron+and+trnL-F+intergenic+spacers.">Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers.</a> J. Plant Res. 115, 381-392 (2002).</li><li>Kress, W. J., Wurdack, K. J., Zimmer, E. A., Weigt, L. A., Janzen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+DNA+barcodes+to+identify+flowering+plants.">Use of DNA barcodes to identify flowering plants.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (23), 8369-8374 (2005).</li><li>Roodt-Wilding, R., Spies, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+relationships+in+southern+African+chloridoid+grasses+(Poaceae)+based+on+nuclear+and+chloroplast+sequence+data.">Phylogenetic relationships in southern African chloridoid grasses (Poaceae) based on nuclear and chloroplast sequence data.</a> Systematics and Biodiversity. 4, 401-415 (2006).</li><li>Brody, J. R., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+boric+acid:+a+Tris-free,+cooler+conductive+medium+for+DNA+electrophoresis.">Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.</a> BioTechniques. 36, 214-216 (2004).</li><li>Chou, C. -. H., Tsai, C. C. <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+in+the+intergenic+spacer+of+ribosomal+DNA+of+Imperata+cylindrica+(L.)+Beauv.+var.+major+(Cogongrass)+populations+in+Taiwan.">Genetic variation in the intergenic spacer of ribosomal DNA of Imperata cylindrica (L.) Beauv. var. major (Cogongrass) populations in Taiwan.</a> Botanical Bulletin of Academia Sinica (Taipei). 40, 319-332 (1999).</li></ol>

我们什么都没有透露。

<html>美国苗圃和景观行业的蓬勃发展，培育和销售具有异国情调和新的植物物种。这一点，再加上贸易的日益全球化，增加了机会，将进入一个外来的植物物种，建立，在美国传播的能力依赖于信息往往是不提供的，包括可能成为侵入联邦监管等植物，正确的分类，并从本地和归化类群的遗传特异性。因为我们的外来入侵植物的知识往往是有限的，隐藏的入侵特征的进口植物已心甘情愿地介绍了学习后，他们侵略我们的农业和自然资源。该协议旨在解决一，与有关的问题白茅提供的第一个简化的分子生物学方法，可以准确区分野生型的茅草和恢复其观赏JBG对口品种。 jove_content“对于本协议的发展&gt;，野生型的茅草收集池河林业单位在2008年6月在圣罗莎县附近周杰伦，FL归人口。JB​​G从商业幼儿园（蓝鸟苗圃，公司采购。）在2008年6月以及从屋主在哥伦比亚集合，莫JBG恢复于2008年6月分别在克莱姆森大学，资深大律师的坎贝尔地质博物馆的院子里得到;从2009年6月的大学在河谷，马公园;和在哥伦比亚，2009年莫（由利兰Cseke确定）房主的前院。所有工厂均保持在阿拉巴马大学（汉茨维尔，AL）位于一个温室。 从这些植物中采集的DNA基因测序，包括常用条码植物2 9个独立的DNA地区进行深入的比较。在所有情况下，JBG序列分别为100％匹配JBG恢复，从而有助于验证，JBG确实恢复到一个绿色，微创的形式。唯一的核ITS和叶绿体 trnL - F地区有差异，可用于基因区分茅草和JBG。 ITS区，总共有一间茅草和JBG 3个SNPs（单核苷酸多态性），而trnL-F区有2个SNP位点和2个InDel（插入和删除）。这些遗传差异允许多种特定的PCR引物进行开发，可以区分野生型的茅草和JBG恢复。最可靠的结果都来自来自叶绿体trnL-F区的引物。因此，这个协议的基础上的茅草，JBG叶绿体基因组trnL - F地区和恢复JBG（ 图4）之间的序列差异。 为了帮助产生更具体的问题品种的引物，并帮助人密切相关的物种，避免误报升称为trnL-F序列的一白茅品种从相关草种（29种43个独立的序列， 例如 ， 香茅citratus，Sorghastrum incompletum，薏苡薏苡，芒草，甘蔗，假高粱）trnL-F序列进行了比较。虽然，我们研究了各种特异引物序列，全国29个种的草，各种特异引物的特异性尚未全面审查能力的DNA扩增大多数其他草种。因此，应仔细辨认用于DNA提取的组织， 因为 ...... 白茅之前，要启动该协议。如果基层不能被认定为茅草或JBG，那么我们建议测序的PCR产物，以确保序列完全匹配，以茅草或JBG。目前，最准确的方法来验证一个给定的草标本的身份是执行上的 T PCRRNL-F和ITS区序列验证和比较准确识别类群已知序列的序列PCR产物。 DNA可扩增引物，在此协议详细（trnL-F区）或其他引物，可在13-15其他出版物。测序是更多的劳动力和成本比使用简化程序密集。 用于PCR的引物的质量是手术成功的关键。我们已在此过程中，从斜生物公司（引物<a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> ，汉茨维尔，AL）。订购从斜生物的引物的优点是，他们储存大量的底漆生产作为我们优化我们的实验室中使用的引物序列相同的样本，每个等分。因此，引物，不只能有相同的序列，但嘿来自完全相同的生产批号，在此协议。使用来自同一地段的引物，可以帮助避免无关的变量，在过程中可能产生的PCR引物的质量差异。同样的，而其他的Taq聚合酶应该罚款PCR技术，使用的Taq聚合酶的质量会影响PCR结果的质量。为了让更好的PCR试剂的一致性，我们优化的协议，自Clontech的试剂。的优势2聚合酶（Clontech公司，加州Mountain View，猫＃639201或639202）是一个强大的，热启动Taq酶的混合物和校对酶，有助于提供高特异性和更准确的扩增。 因为这个协议为依托，叶绿体DNA，这是母系继承草茅草和JBG基因型之间的杂交事件可能不会被捕获我们的分子鉴定程序。在情况hypridization是suspe中反恐执行局，我们建议使用核武器地区，是从父母双方继承。最常用的非质的考虑在植物基因分型的可变区是核核糖体ITS区13-15,17。目前，我们正在朝着复核及其在同一PCR管区 ​​扩增叶绿体trnL-F区的进展情况。复质与核DNA区域可能会规避使用单独的局限性，但这种方法需要优化和额外的评估，以确定在逐案的可行性。使用实时定量PCR（定量PCR）和新技术，如分子信标（荧光引物探针），也被评为故障安全和准确的植物基因分型方法。 这里介绍的协议提供了一个快速和可靠的方法来区分JBG恢复野生型茅草。我们鼓励使用此协议的RS与我们联系，报告使用此协议所得的结果。这种共享的信息将有助于提供对JBG恢复的分布信息。这也将有助于在美国农业部的监管作出明智的决策，行动可能需要规避的高度侵袭性​​的茅草品种的扩散和潜在的杂交。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DNeasy植物迷你套件</td><td> Qiagen公司，瓦伦西亚，CA </td><td> 69104或69106 </td><td>任何有信誉的基因组植物DNA试剂盒，得到质量较好的DNA，应该为这些程序正常工作。 </td></tr><tr><td> trnF基因（GAA） - F </td><td>斜生物公司</td><td> 3-0578 </td><td>正向积极的控制底漆</td></tr><tr><td> trnL（5&#39;外显子） - C </td><td>斜生物公司</td><td> 3-0579 </td><td>反向阳性对照引物</td></tr><tr><td> trnLF-C F1 </td><td>斜生物公司</td><td> 3-0864 </td><td>前进野生型的茅草底漆</td></tr><tr><td> trnLF-C - R1 </td><td>斜生物公司</td><td> 3-0865 </td><td>逆转野生型的茅草底漆</td></tr><tr><td> trnLF R-F2 </td><td>斜生物公司</td><td> 3-0866 </td><td>向前JBG和JBG恢复底漆</td></tr><tr><td> trnLF-R - R2 </td><td>斜生物公司</td><td> 3-0867 </td><td>扭转JBG和JBG恢复底漆</td></tr><tr><td>优势超纯PCR dNTP混合液</td><td> Clontech公司，加州Mountain View </td><td> 639125 </td><td></td></tr><tr><td>优势2聚合酶</td><td> Clontech公司，加州Mountain View </td><td> 639201或639202 </td><td>一个好的校对，热启动Taq酶</td></tr></tbody></table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

野生I 型白茅 （茅草）是世界十大最严重的入侵植物之一，在整个非洲，亚洲，欧洲，新西兰，大洋洲和美洲1-2在73个不同国家的农业和自然资源造成负面影响。茅草形成迅速蔓延，单优势的看台上，取代了原生植物物种种类繁多，反过来威胁流离失所牧草和住所的原生植物物种赖以生存的本土动物。添加到这个问题，观赏各种[ 一白茅VAR。koenigii（Retzius）被广泛销售下白茅 &#39;赤芍，红男爵，日本血草（JBG）的名称。该品种是公认的无菌，无创，被认为是理想的观赏为红色的叶子。然而，正确的条件下，JBG可以产生可行的种子（卡罗尔Holko，2009年个人通信），可以恢复到一个绿色的我nvasive形式往往是茅草区分野生型入侵品种4（ 图1）的显着特点。这使得识别使用形态，即使是训练有素的植物分类学家的一项艰巨的任务。 JBG一个积极的绿色环保型的逆转也并非罕见。利用核和叶绿体DNA编码和可变区序列的比较，我们已经证实，JBG内，南卡罗来纳州，马​​里兰州和密苏里州已恢复到绿色入侵。 JBG已出售和种植在美国大陆，那里是不活跃的茅草为患只是每个国家。恢复问题不能很好地理解的程度，因为恢复的植物是无证，经常遭到破坏。 这种分子的协议中的应用提供了一个方法来确定JBG恢复，可以帮助保持这些品种的共同发生1ND可能杂交。茅草是一个的预留outcrosser，越过了不同基因型时，可以产生可行的风力分散种子遍布茅草5-7宽的距离。 JBG略有不同基因型比茅草和可能能够形成茅草可行的杂交种。添加到这个问题，JBG是越冷，比茅草8-10树荫宽容，这两个品种之间的基因流可能产生更积极的杂交种，是树荫宽容，比野生型的茅草和冷耐寒。而野生型的茅草目前滋生全球超过490万公顷，在美国东南部，它滋生超过50万公顷，是能够占据大部分的美国，因为它迅速蔓延北上，由于其广泛的利基和地理潜在3,7,11。潜在的遗传十字路口的美国农业部动植物检疫局联邦有毒本周“节目的严重关切。目前，美国农业部动植物检疫局禁止在国家WH JBGERE有茅草主要虫害（例如，佛罗里达州，阿拉巴马州，密西西比州）。然而，预防相结合的两个品种可以证明白茅和JBG扩大其分布更加困难。此外，JBG恢复的分布是目前未知的，没有识别能力，通过这些品种形态，一些茅草虫害可能JBG恢复的结果。不幸的是，目前的分子生物学方法鉴定通常依靠的AFLP（扩增片段长度多态性）和DNA测序，这两者都是费时又费钱。在这里，我们提出的第一个符合成本效益和可靠的基于PCR的分子基因分型方法准确区分茅草和JBG恢复。

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

我们提供1成本效益和快速的分子分型的协议，采用各种特定的PCR引物，目标内的叶绿体trnL - F间隔区DNA序列差异区分品种<em&gt;白茅</em&gt;（茅草）不能被单独区分形态。这些品种包括联邦政府列出的有害杂草，白茅和密切相关的，广泛的观赏品种，我<em&gt;。白茅</em&gt; VAR。<em&gt; koenigii</em&gt;（日本血草）。

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

a-pcr-based-genotyping-method-to-distinguish-between-wild-type

Research

JoVE Journal

Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

31.4K 次观看.  The University of Alabama, Huntsville. 我们提供1成本效益和快速的分子分型的协议，采用各种特定的PCR引物，目标内的叶绿体trnL - F间隔区DNA序列差异区分品种白茅（茅草）不能被单独区分形态。这些品种包括联邦政府列出的有害杂草，白茅和密切相关的，广泛的观赏品种，我。白茅 VAR。 koenigii（日本血草）。

视频: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

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 ...

科研

生物学

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

蛋白膜覆盖含量：一个协议测试可溶性和不溶性蛋白质之间的相互作用在体外</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

优化的基于PCR的检测支原体

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽：为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

基因分型的植物和动物样品，恕不另行DNA的纯化

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

测定野生型肝脏和牛奶样品中的唾液酸<em&gt; CMAH</em&gt;敲出小鼠

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...