拟南芥的样品和有关PCR引物，我们感谢的JaakkoKangasjärvi教授和他的研究小组，的植物逆境集团，植物分子生物学，生物科学系，赫尔辛基大学。与传统方法的实验进行了夫人爱里Lamminmäki。 转基因小鼠提供样品，生物医学/生物化学研究所，赫尔辛基大学的博士JAANA Vesterinen。 哈里斯Uni-CORE核心和哈里斯切割垫的Shunderson通信公司的商标，所有其他商标的财产的Thermo Fisher Scientific公司及其附属公司的商标或注册商标。

1。 拟南芥植物个体基因分型与直接议定书<ol><li>要开始dCAPS特异性的基因分型检测，直接在拟南芥的植物叶，首先制订20或50μl的PCR反应使用的Phire的植物直接PCR试 ​​剂盒如表1中描述的。 </li><li>其次，从拟南芥叶以Harris Uni-CORE核心和哈里斯切割垫削减了0.50毫米打孔。牢牢握住冲床，按切削刃的组织和穿孔机来回旋转。 </li><li>按柱塞喷射冲头的PCR反应混合物的光盘插入。确保样本落入PCR溶液，不会粘到管壁。 </li><li>清洁的前沿之间的冲床，每个样品到2％次氯酸钠溶液浸渍，以防止交叉污染。按柱塞上下几次，用干净的纸巾擦拭的最前沿。切割垫也应该样品之间的冲洗。 </li><li>其次，采用了Thermo Scientific的PIKO的24孔热循环仪和赛默飞世尔科技PIKO PCR板进行PCR反应，使用表2中描述的循环条件。也可以在传统的PCR热循环仪进行PCR反应。 </li><li> PCR后，自旋向下的植物材料。 5微升上层清液转移到新的微量离心管中。加入4μl的水和1微升限制性内切酶，引入SspI。轻轻混匀后短暂离心管的底部收集内容。孵育一小时将反应混合物在37℃下的限制性内切酶失活，在65℃下孵育20分钟。 </li><li>当扩增的DNA直接从植物组织中，将PCR产物包括厂房及PCR衍生的组成部分，可能会干扰用限制性消化酶。因此，它可能是必要的，以稀释（ 例如，在水中的1:2或1:3），或纯化的PCR产物之前吨他随后的消化，例如，通过使用一个合适的商业化试剂盒如Thermo Scientific GeneJET PCR Purification Kit纯化。 </li><li>限制性内切酶消化后，得到的片段在琼脂糖凝胶上进行分析。 2.5微升5×加样缓冲液加入到反应，并进行琼脂糖凝胶电泳所得到的混合物与10μl。 </li></ol> 2。从15岁的老橡树叶扩增特定DNA片段用稀释协议<ol><li>开始的橡树叶，切成2毫米的打孔。 </li><li>将样品的的Phire电厂直接PCR试剂盒提供的稀释液为20μL。清洁尖端的冲床和切割垫和以前一样。 </li><li>叶样品粉碎，用100微升移液器吸头按它简单地对管壁。根据不同的植物材料，稀释协议有时效果更好而不破碎的样本。 </li><li>短暂地旋转样品中微量直到t他的反应是在管的底部。 </li><li>将上清液可以直接使用，在PCR中，或者它可以稀释在无菌水中1:10或1:100取决于关于样品类型。使用0.5μl到1微升上清液或稀释作为一个20微升的PCR反应的模板。准备如表1中所述的反应。 </li><li>一旦反应的准备，采用了Thermo Scientific的PIKO的24孔热循环仪和赛默飞世尔科技PIKO PCR板进行PCR反应，使用表2中描述的循环条件。也可以在传统的PCR热循环仪进行PCR反应。 </li><li>在PCR后，对反应中添加5μl的5×加样缓冲液，并用15μl的所得到的混合物进行琼脂糖凝胶电泳。 </li></ol> 3。使用稀释协议的转基因小鼠的基因分型<ol><li>转基因小鼠放置一个2毫米的打孔的M开始稀释的协议乌斯含0.5μL的DNARelease添加剂提供，与Phire动物组织直接PCR试剂盒的稀释液在20μl的耳朵。 </li><li>样品在室温下孵育2分钟，然后在98℃下孵育2分钟</li><li>在微量短暂旋转样品，直到反应是在管的底部。 </li><li>使用1μl的上清液为20微升的PCR反应中，作为模板，如表3中所述的方法制备。任何上清液是不被马上使用，可以被转移到新的试管中，并储存于-20℃下</li><li>其次，采用了Thermo Scientific的PIKO的24孔热循环仪和赛默飞世尔科技PIKO PCR板进行PCR反应，使用表4中所述的循环条件。同样，也可以进行PCR反应，在传统的PCR循环仪。 </li><li>进行PCR，对反应中添加5μl的5×加样缓冲液，并进行琼脂糖凝胶电泳瓦特第i个15微升所得的混合物。 </li></ol> 4。代表性的成果在dCAPS特异性技术的限制性位点内的SNP的利益要么是使用与一个或多个错配的靶DNA的PCR引物引入或销毁。直接从叶拳dCAPS特异性技术的拟南芥植物个体进行基因分型。的扩增产物进行消化，并揭示每个个体的基因型产生的碎片，用凝胶电泳分析（ 图2）。 的160 bp的PCR产物在拟南芥基因组中含有景点的SNP位点（G或A等位基因）在这个例子中，从植物叶冲头与Phire电厂直接PCR试 ​​剂盒进行扩增。的正向引物在3&#39;端包含一个不匹配，创建一个SspI特定的限制位点中的目标DNA，其中包括的利益的SNP（A等位基因），但不在其他等位基因的SNP（ 图2B）。 稀释协议是必要的具有挑战性的植物材料，如橡树叶，由于其高浓度的酚类化合物。用这里描述的稀释协议，297 bp的叶绿体的DNA片段的扩增使用3步骤的协议。橡树叶标本与不同稀释度的图3中所示，除了对阳性和阴性对照。 Phire动物组织直接PCR试剂盒采用一个具有挑战性的协议，其中只有一个引物组扩增两个片段被用于具有大尺寸的差异，导致在丰富的产量为1,500 bp的转基因小鼠和200 bp的正确尺寸的产品野生型小鼠（ 图4）。杂合子小鼠较弱的上部带是由于这两个模板（等位基因）的相同的引物对的竞争，但是，genotypING的结果是明确的。 使用稀释协议制备的样品的稳定性进行了测试。结果表明，稀释样品，可以存储在-20℃下为至少一年。此外，反复冷冻/解冻循环没有显着影响的反应（ 图5）。还示出的是稳健的扩增使用Phire DNA聚合酶和热启动 Taq聚合酶和纯化的DNA（ 图6）相比的直接PCR法从小鼠耳组织样本。 <table border="1"><tbody><tr><td fo:width="2in"> 组件 </td><td fo:width=".75in"> 20μl反应 </td><td fo:width=".75in"> 50μl反应体系 </td><td fo:width=".75in"> 终浓度。 </td></tr><tr><td> H 2 O </td><td>加至20微升</td><td>添加到50微升</td><td></td></tr><tr><td> 2个Phire植物PCR缓冲液</td><td> 10微升</td><td> 25微升</td><td> 1个</td></tr><tr><td>引物A </td><td>所述微升</td><td>所述微升</td><td> 0.5μM </td></tr><tr><td>引物B </td><td>所述微升</td><td>所述微升</td><td> 0.5μM </td></tr><tr><td> Phire热启动II DNA聚合酶</td><td> 0.4μL </td><td> 1微升</td><td></td></tr><tr><td>采样/直接协议</td><td></td><td> 0.50 mm打孔</td><td></td></tr><tr><td>采样/稀释协议</td><td> 0.5-1微升</td><td></td><td></td></tr></tbody></table> 表1中。<html>在本协议中使用的植物PCR反应条件。 <table border="1"><tbody><tr><td fo:width="1in"> 循环步骤 </td><td fo:width=".5in"> 温度。 </td><td fo:width=".5in"> 时间 </td><td fo:width=".4in"> 循环 </td></tr><tr><td>初始变性</td><td> 98°C </td><td> 5分钟</td><td> 1 </td></tr><tr><td>变性 退火* 延期</td><td> 98°C X°C 72°C </td><td> 5秒 5秒 20秒</td><td> 40 </td></tr><tr><td>最后延伸</td><td> 72°C 4°C </td><td> 1分钟 持有</td><td> 1 </td></tr></tbody></table> 表2。植物PCR循环条件中使用此协议。 * TM计算器oscientific.com机构/ pcrwebtools“目标=”_blank“&gt; www.thermoscientific.com / pcrwebtools的建议当确定要使用Phire热启动II DNA聚合酶的引物的Tm。推荐的引物的退火温度≤20 nt是等于较低的Tm给出由webtool。对于引物&gt; 20 nt的，使用的退火温度3℃高于较低的Tm由webtool给出。 <table border="1"><tbody><tr><td fo:width="2in"> 组件 </td><td fo:width=".75in"> 20μl反应 </td><td fo:width=".75in"> 终浓度。 </td></tr><tr><td> H 2 O </td><td>加至20微升</td><td></td></tr><tr><td> 2个Phire动物组织PCR缓冲液</td><td> 10微升</td><td> 1个</td></tr><tr><td>引物A </td><td>所述微升</td><td> 0.5μM </td></tr><tr><td>引物B </td><td>所述微升</td><td> 0.5μM </td></tr><tr><td> pH值愤怒热启动II DNA聚合酶</td><td> 0.4μL </td><td></td></tr><tr><td>采样/稀释协议</td><td> 1微升</td><td></td></tr></tbody></table> 表3。在本协议中使用的动物组织的PCR反应条件。 <table border="1"><tbody><tr><td fo:width="1in"> 循环步骤 </td><td fo:width=".5in"> 温度。 </td><td fo:width=".5in"> 时间 </td><td fo:width=".4in"> 循环 </td></tr><tr><td>初始变性</td><td> 98°C </td><td> 5分钟</td><td> 1 </td></tr><tr><td>变性 退火* 延期 </td><td> 98°C X°C 72°C </td><td> 5秒 5秒 20秒≤1 KB 20秒/ KB 1 KB </td><td> 40 </td></tr><tr><td>最后延伸</td><td>72°C 4°C </td><td> 1分钟 持有</td><td> 1 </td></tr></tbody></table> 表4。在本协议中使用的动物组织PCR循环条件。 *的Tm计算器时，建议在<a href="http://www.thermoscientific.com/pcrwebtools" target="_blank">www.thermoscientific.com / pcrwebtools的</a>决定使用Phire热启动II DNA聚合酶的引物的Tm。推荐的≤20核苷酸的引物的退火温度是等于较低的Tm由webtool给出。对于引物&gt; 20 nt的，使用的退火温度3℃，高于给定的较低的Tm由webtool。 <img src="/files/ftp_upload/3844/3844fig1.jpg" alt="图1" /> 图1。直接PCR的工作流程。，直接PCR可以进行使用两个替代协议。在直接协议中一个微小的量的样品直接添加到PCŕ反应。稀释协议采用简短的预孵育PCR步骤之前从样品材料释放DNA。 <img src="/files/ftp_upload/3844/3844fig2.jpg" alt="图2" /> 图2。 拟南芥植物个体的基因分型与dCAPS特异性技术直接从叶冲头。 A）图2A示出的原则进行的dCAPS特异性检测。内SNP的利益被引入的限制性位点，通过PCR使用的引物与一个或多个的目标DNA的错配或移除。对PCR产物进行消化，所得片段揭示每个个体的基因型时，通过电泳分析。B）从植物叶片0.50毫米冲头被直接放置在50μl的PCR反应。引入一个独特的SspI位点的A等位基因的引物进行扩增的160 bp的PCR产物含有的SNP位点（G或A等位基因）。未纯化的PCR产物酶切特德用SspI，限制性内切酶。所得片段用3％的琼脂糖凝胶电泳分析。泳道M是大小标记物，泳道A和G对应于每个样品的SNP等位基因。提取DNA，然后通过PCR和酶切（数据未示出）通过常规的分析，将所得的结果证实。无模板DNA的阴性对照反应，包括在测试设置，并运行一个单独的琼脂糖凝胶上进行消化（数据未示出）之前，与实际的PCR反应平行。 <img src="/files/ftp_upload/3844/3844fig3.jpg" alt="图3" fo:content-width="5.5in" fo:src="/files/ftp_upload/3844/3844fig3highres.jpg" /> 图3。用橡树叶样品稀释协议。稀释协议被用来扩增的297 bp的DNA片段，叶绿体DNA在橡树叶样品（3步协议，退火温度为62°C），不同的稀释液（1:100，1:10， ：1和2:1），阳性和阴性对照操纵，[C +：阳性对照纯化的DNA（ 拟南芥 ），C-：没有模板DNA的阴性对照。代表样品的采集样品命名网站在芬兰（镇）和样品编号。 <img src="/files/ftp_upload/3844/3844fig4.jpg" alt="图4" /> 图4。基因分型的转基因小鼠的一个引物对使用的稀释协议。九个个别小鼠耳组织中（泳道1-9）被放置入20μl的稀释缓冲液包括DNARelease添加剂。经过温育在室温下和98℃，1微升上清液用作20微升的PCR反应中的模板。片段的大小：1,500基点（转基因）和200bp（野生型）。 <img src="/files/ftp_upload/3844/3844fig5.jpg" alt="图5" /> 图5。制备的样品的稀释协议是稳定的，长期贮存。小鼠耳组织样品公司<html>在20μl的稀释缓冲液包括DNARelease添加剂ubated进行反复冷冻/解冻（泳道1），在-20℃下存储一年（泳道2），或立即用于PCR（泳道3）。扩增片段大小为900 bp的，1500 bp和3200 bp的。 <img src="/files/ftp_upload/3844/3844fig6.jpg" alt="图6" /> 图6。 Phire动物组织直接PCR试 ​​剂盒优于热启动Taq DNA聚合酶结合的商业DNA纯化系统。小鼠耳组织，用稀释协议Phire动物组织直接PCR试 ​​剂盒扩增，扩增子（0.5-1.5）。为了进行比较，DNA首先被使用的是商业的DNA提取试剂盒纯化和相同的片段，根据制造商的建议使用一个热启动Taq DNA聚合酶扩增。只有，与Phire动物组织直接PCR试剂盒均全部扩增子成功地扩增。 T“&gt; <img src="/files/ftp_upload/3844/3844fig7.jpg" alt="图7" /> 图7。通用的工作流程比较估计次/消耗的试剂。Thermo Scientific的直接PCR方法相比，DNA的提取方法，提取缓冲液的方法，PCR缓冲方法。每种方法之前，PCR步骤中列出的红色文字的估计时间。 Thermo Scientific能够直接PCR方法之前PCR样品制备只需要0-4分钟。下面的每一种方法中，所消耗的试剂被列出。 Thermo Scientific能够直接PCR方法中的其它方法相比，使用最少的试剂。 <a href="http://www.jove.com/files/ftp_upload/3844/3844fig7large.jpg" target="_blank">点击此处查看大图</a> 。

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	<li>Doyle, J. J., Doyle, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+DNA+isolation+procedure+for+small+quantities+of+fresh+leaf+tissue.">A rapid DNA isolation procedure for small quantities of fresh leaf tissue.</a> Phytochemical Bulletin. 19, 11-15 (1987).</li><li>Kim, C. S., Lee, C. H., Shin, J. S., Chung, Y. S., Hyung, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+isolation+of+high+quality+genomic+DNA+from+fruit+trees+and+conifers+using+PVP.">A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP.</a> Nucleic Acids Research. 25, 1085-1086 (1997).</li><li>Neff, M. M., Neff, J. D., Chory, J., Pepper, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dCAPS,+a+simple+technique+for+the+genetic+analysis+of+single+nucleotide+polymorphisms:+experimental+applications+in+Arabidopsis+thaliana+genetics.">dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.</a> The Plant Journal. 14, 387-392 (1998).</li><li>Michaels, S. D., Amasino, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+method+for+detecting+single-nucleotide+changes+as+polymorphic+markers+by+PCR.">A robust method for detecting single-nucleotide changes as polymorphic markers by PCR.</a> The Plant Journal. 14, 381-385 (1998).</li><li>John, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+isolation+of+RNA+and+DNA+from+plants+containing+polyphenolics.">An efficient method for isolation of RNA and DNA from plants containing polyphenolics.</a> Nucleic Acids Research. 20, 2381-23 (1992).</li><li>Wang, Z., Storm, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+mouse+tails.">Extraction of DNA from mouse tails.</a> BioTechniques. 41, 410-412 (2006).</li><li>Malumbres, M., Mangues, R., Ferrer, N., Lu, S., Pellicer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+High+Molecular+Weight+DNA+for+Reliable+Genotyping+of+Mice.">Isolation of High Molecular Weight DNA for Reliable Genotyping of Mice.</a> BioTechniques. 22, 1114-1119 (1997).</li><li>Yang, Y., Kim, J. Y., Soh, M. S., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+gene+amplification+from+Arabidopsis+Leaves+Using+AnyDirect+system.">A simple and rapid gene amplification from Arabidopsis Leaves Using AnyDirect system.</a> Journal of Biochemistry and Molecular Biology. 40, 444-447 (2007).</li><li>Sambrook, J., Fritsch, E., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory. , (1989).</li><li>Meldgaard, M., Bollen, P. J. A., Finsen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+method+for+sampling+and+extraction+of+mouse+DNA+for+PCR.">Non-invasive method for sampling and extraction of mouse DNA for PCR.</a> Laboratory Animals. 3, 413-441 (2004).</li></ol>

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这里展示的直接PCR方法允许直接从少量未纯化的样品进行PCR扩增，减少所需要的时间，并简化为植物和动物的基因分型（ 图7）的工作流程。此处也表现出与直接PCR的方法（ 图3）是使用的稀释组合协议。困难的样品（ 如老化植物的叶子，植物，含有干扰物质）或长（或富含GC），扩增产物稀释协议。时，开始一个新的直接PCR实验中，允许反应优化的，该协议是特别有用的。该协议可以作为一种工具来解决偶尔的困难，例如低的产品产率，由于在组织材料和/或使用的引物的差异。此外，运行多个反应时，从相同的样品，使用的稀释协议确保将整个样品在一个没有被消耗反应。 为了净化从植物中用于PCR的DNA，该公约来进行细胞裂解，然后由DNA分离使用各种部件，有可能会干扰PCR分析8。对于特别具有挑战性的植物与酚含量高，另外聚乙烯吡咯烷酮，传统上被用于删除与DNA结合的化合物，细胞裂解后的2,5。这里可以明显看出，这些复杂的，时间密集的步骤，可避免通过使用的Phire植物直接PCR试剂盒来检测目标DNA。使用的敏感dCAPS特异性技术直接从拟南芥的植物的叶子（ 图2）的细胞裂解和DNA分离步骤可以省略。这里表明，稀释协议有效地管理问题的组件，可以干扰用聚合酶链反应（ 图3）的存在下。 传统上，动物基因分型的先决条件是isolati通过一种有毒的，耗费时间的过程，其特征在于由一个漫长的孵化用蛋白酶K消化皮肤和结缔组织，其次通过有机溶剂提取，乙醇沉淀，并离心步骤6,9,10从动物组织中的基因组DNA上。这里表明，通过使用的Phire动物组织直接PCR试 ​​剂盒来实现这个过程的简化，允许转基因小鼠进行基因分型，恕不另行DNA纯化（ 图4）。在本文中演示的例子是特别具有挑战性，因为采用了大尺寸的差异，只有一个用于两个片段的扩增引物组。尽管协议的先天困难，使用直接PCR议定书简单稀释导致的两个PCR产物（ 图4）的高收率的方法配合使用。 PCR为基础的目标DNA检测，有很多的应用研究，包括基因型分析，以确定发展，生理和疾病基因的角色。由于专门的DNA聚合酶抑制剂的耐受性，该协议可以在最短的时间，恕不另行DNA纯化完成。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td>公司</td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Phire植物直接PCR试剂盒</td><td>赛默飞世尔科技</td><td> F-130 </td><td> 200次反应（50微升） </td></tr><tr><td> Phire动物组织直接PCR试剂盒</td><td>赛默飞世尔科技</td><td> F-140 </td><td> 200 reations（各50微升） </td></tr><tr><td>哈里斯单核0.35毫米（粉红色） </td><td>赛默飞世尔科技</td><td> F-180S / L </td><td>数量：5/25 </td></tr><tr><td>哈里斯单核0.50毫米（蓝色） </td><td>赛默飞世尔科技</td><td> F-185S / L </td><td>数量：5/25 </td></tr><tr><td>哈里斯切割垫6.4×7.6厘米</td><td>赛默飞世尔科技</td><td> F-190 </td><td>数量：5 </td></tr><tr><td> SSPI </td><td>赛默飞世尔科技</td><td> ER0771 </td><td>加入100μl（100次反应） </td></tr><tr><td> PIKO的24孔热循环仪</td><td>赛默飞世尔科技</td><td> TCP0024 </td><td></td></tr><tr><td> 24孔PIKO PCR板</td><td>赛默飞世尔科技</td><td> SLP0241 </td><td></td></tr><tr><td> GeneJET PCR 纯化试剂盒</td><td>赛默飞世尔科技</td><td> K0701 K0702 </td><td> 50税号 250税号</td></tr></tbody></table>

genotyping of plant and animal samples without prior dna purification

的直接PCR方法有利于PCR扩增直接从少量未纯化的样品，这里用于表明几种植物和动物组织（ 图1）。直接PCR的基础上特别设计的赛默飞世尔科技Phusion Phire DNA聚合酶，其中包括双链DNA结合结构域，使他们独特的性能，如高耐受性抑制剂。 PCR为基础的目标DNA检测在植物研究中有许多应用，包括转基因植株基因型分析和验证。传统的PCR方法从植物组织中涉及的初始DNA分离步骤，这可能需要昂贵的或有毒的试剂。该过程是耗时的，并增加1，2的交叉污染的风险。相反，通过使用Thermo Scientific的Phire植物直接PCR试剂盒目标DNA可以被容易地检测到，没有现有的DNA提取。在该模型中表现出在这里，例如来自裂解的扩增多态性序列分析（dCAPS特异性）3,4进行直接从拟南芥的植物的叶子。可以使用dCAPS特异性基因分型分析，以确定由SNP等位基因特异的限制性核酸内切酶消化3的单核苷酸多态性（SNP）。 某些植物样品往往是更具挑战性的，当使用直接PCR方法，因为它们含有干扰PCR的组件，如酚类化合物。在这些情况下，需要一个额外的步骤，以除去该化合物是传统2,5。在这里，此问题被克服，通过使用一个快速和容易的稀释随后通过直接PCR扩增（ 图1）的协议。作为一个具有挑战性的植物模型的样本含有大量的酚类物质，包括单宁酸，十五年的老橡树叶。 进行基因转移到小鼠中被广泛用于在开发与研究的作用的基因pment，生理与人类疾病。这些动物的使用需要筛选的转基因的存在，通常用PCR。传统上，这涉及一个耗时的DNA分离步骤中，在此期间，用于PCR分析的DNA纯化从耳，尾或脚趾组织6,7。然而，赛默飞世尔科技Phire动物组织直接PCR试剂盒的转基因小鼠进行基因分型，恕不另行DNA纯化。实现此协议中的转基因小鼠的基因分型的直接从小鼠耳组织，如这里用于显示其中只有一个引物组，用于扩增两个片段的大小差别很大的一个挑战性例子。

Watch this Scientific Journal Video about Genotyping of Plant and Animal Samples without Prior DNA Purification at JoVE.com

无需事先纯化 DNA 即可对植物和动物样品进行基因分型 |生物学 |视频

Genotyping of Plant and Animal Samples without Prior DNA Purification

这里提出的直接PCR方法便于直接从少量的未精制的植物和动物组织的PCR扩增。

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 ...

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31.0K 次观看.  Thermo Fisher Scientific. 这里提出的直接PCR方法便于直接从少量的未精制的植物和动物组织的PCR扩增。

视频: Genotyping of Plant and Animal Samples without Prior DNA Purification

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

从原核文化精简纯化的质粒DNA

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

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

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

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 ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

从复杂的动物相关样品测序宏基因组和Metatranscriptomes：净化不纯

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

扫描电子显微镜（SEM）协议有问题的工厂，卵菌和真菌样本

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

无需细胞分选即可从快速冷冻的异质组织样品中分离细胞类型特异性RNA

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...