感谢上海托洛生物科技在开发C-Brick标准方面的技术援助。这项工作得到了中国科学院战略重点研究计划（批准号:XDB19040200）的资助。

1.制备crRNA <ol><li> 制备crRNA模板 <ol><li>将无RNase的水中的单个寡核苷酸（ 表1 ）重新悬浮至10μM的浓度。 </li><li>加入22.5μL顶链寡核苷酸（ 表1中 T7-F），22.5μL底链寡核苷酸（ 表1 ）和5μL10x退火缓冲液至0.2mL PCR管。确保总体积为50μL。 注意: 表1中显示了六种不同的底链寡核苷酸，每种寡核苷酸应与顶链T7-F单独配对。 表1中的小写字母表示要转录为crRNA指导序列的序列。可以使用10x Taq PCR缓冲液代替10x退火缓冲液。 </li><li>将PCR管放入热循环仪中。 </li><li>执行退火程序:95℃下初始变性5分钟，然后冷却从95-20℃，热循环仪每分钟降低1℃。 注意:立即使用样品进行步骤1.2.1。 </li></ol></li><li> 转录和纯化的crRNA <ol><li>加入38μL无RNase的水，8μL来自步骤1.1.4的模板，20μL5x T7转录缓冲液，20μLNTP混合物（10μM），4μL重组RNA酶抑制剂（RRI）和10μl μLT7 RNA聚合酶到1.5 mL微量离心管中。确保总体积为100μL。 </li><li>将微量离心管放入37°C水浴过夜（约16 h）。 </li><li>使用RNA清除和浓缩试剂盒来纯化转录的RNA;遵循制造商的协议。 </li><li>用UV-Vis分光光度计定量RNA，并将RNA稀释至10μM浓度。立即使用样品或将其储存在-80°C。 注意:crRNA序列s列于表2 。 </li></ol></li></ol> 2. C-Brick零件的构造（ 即将生物零件插入C砖标准矢量） 注意:此步骤有三个子步骤。对于短生物部分（ 如启动子和终止子），最好使用直接PCR方法将生物部位插入C-Brick标准载体4 。整个矢量序列显示在补充数据中，前缀和后缀序列如图1所示 （下面的步骤2.1）。对于可以通过PCR容易获得的生物部位（ 例如，使用基因组DNA，质粒或从头合成的DNA序列的模板），最好使用无缝组装方法将生物部位插入C-Brick标准载体（下面的步骤2.2）。对于从BioBrick和BglBrick标准获得的那些部件，限制酶介导的消化和T4 DNA连接酶介导的连接可用于将生物部位插入C-Brick标准载体（下文的步骤2.3）。 <ol><li> 通过直接PCR扩增构建 <ol><li>设计和订购PCR扩增的寡核苷酸。 注意:寡核苷酸的5&#39;末端含有DNA部分的序列，寡核苷酸的3&#39;末端含有载体序列（ 即，前向3&#39;末端的"GGATCCACTAGTCTCTAGCTCG"和"GGATCCTTTCTCCTCTTTCTAG"在反向链中）。可以在寡核苷酸的5&#39;端设计无突出端或〜20-bp突出端。具体的例子可以在以前的研究中找到。 </li><li>将超纯水中的单个寡核苷酸重新悬浮至10μM的浓度。 </li><li>在0.2 mL PCR管中，在冰上设置PCR反应:加入18μL超纯水，25μL2x PCR缓冲液，1μLdNTP（10μM），2.5μL来自步骤2.1.2的正向和反向引物（10μM），0.5μL作为模板的C-Brick标准载体（50ng）和0.5μL的高保真DNA聚合酶。确保总体积为50μL。 </li><li>将管直接放入热循环仪中，启动热循环仪程序。立即使用样品或将其冷冻保存在-20°C。 </li><li>热循环程序:98℃一个循环3分钟; 98℃30秒，55℃20秒，72℃2秒;在72℃下循环10分钟。将样品保持在16°C。 </li><li>将9μL步骤2.1.4的混合物加入到1.5 mL微量离心管中。 </li><li>加入1μLDpnI，并在37°C水浴中孵育1 h。 注意:如果引物没有重叠序列，加入0.5μLT4多核苷酸激酶（PNK）和0.5μLT4 DNA连接酶，并孵育22°C水浴1 h。否则，如果引物含有〜20nt的重叠序列，则将DpnI处理的产物直接转化到大肠杆菌 （DH10B）感受态细胞中（步骤2.4）。线性化PCR产物将通过宿主中的重组系统进行环化。 </li><li>立即使用样品或将其储存在-20°C。 </li></ol></li><li> 通过无缝组装施工 <ol><li>设计和订购PCR扩增的寡核苷酸。 注意:寡核苷酸的3&#39;末端含有DNA部分的末端序列，寡核苷酸的5&#39;末端包含线性化C-Brick标准载体序列的末端（ 即 "CTAGAAAGAGGAGAAAGGATCC"为5&#39;前端的末端和反义链5&#39;端的"CGAGCTAGAGACTAGTGGATCC"）。具体的例子可以在以前的研究中找到。 </li><li>重新挂起个人在超纯水中的寡核苷酸浓度为10μM。 </li><li>在0.2 mL PCR管中，在冰上设置PCR反应:加入18μL超纯水，25μL2x PCR缓冲液，1μLdNTP（10μM），2.5μL正向和反向引物（10μM ），0.5μL的模板（20-500ng）和0.5μL的高保真DNA聚合酶。确保总体积为50μL。 </li><li>将管直接放入热循环仪中，运行热循环仪程序。立即使用样品或将其冷冻保存在-20°C。 注意:将热循环仪程序设置为:在98℃下一个循环3分钟;在98℃下30秒，55℃20秒（根据引物的退火温度）和在72℃下1分钟（根据生物部分的长度）30个循环;在72℃下循环10分钟。将样品保持在16°C。 </li><li>使用PCR清理系统纯化PCR产物工具箱遵循制造商的协议。 </li><li>用BamHI消化C-Brick标准载体:向微量离心管中加入34μL超纯水，10μL质粒（1μg），5μL10x缓冲液和1μLBamHI。在37℃下在水浴中孵育2小时。 </li><li>使用凝胶清理系统套件净化上述产品;遵循制造商的协议。 </li><li>加入2μL来自步骤2.2.7的线性化载体（50ng），5μL来自步骤2.2.4的片段（200ng），2μL5x无缝组装缓冲液和1μL无缝组装酶的无缝组装酶至1.5 mL微量离心管。确保总体积为10μL。 </li><li>将其放在37°C水浴中30分钟。立即使用样品或将其冷冻保存在-20°C。 </li></ol></li><li> 通过限制酶介导的消化和T4 DNA连接酶介导的连接进行构建。 注意:对于从BioBrick（步骤2.3.4-2.3.7）和BglBrick（步骤2.3.1-2.3.3）标准品获得的那些部分，可以使用限制酶介导的消化和T4 DNA连接酶介导的连接来插入生物部位成为C-Brick标准载体。 <ol><li>用BamHI和BglII对BglBrick标准品进行消化:向微量离心管中加入34μL超纯水，10μL质粒（2μg），5μL10x缓冲液3，0.5μLBamHI和0.5μLBglII。在37°C水浴中孵育2小时。 </li><li>进行1％琼脂糖凝胶电泳，并使用凝胶清理系统试剂盒纯化该片段。 </li><li>将片段和C-Brick标准载体连接:将2μL来自步骤2.1.12的线性化载体（50ng）和6μL来自步骤2.3.3的片段（200ng）加入到1.5mL微量离心管中。加入1μL10 x T4 DNA连接酶缓冲液和1μLT4 DNA连接酶。在22°C孵育2小时。立即使用样品或冻结e并将其储存在-20°C。 </li><li>用XbaI和SpeI对BioBrick标准件进行摘录:向微量离心管中加入34μL超纯水，10μL质粒（2μg），5μL10x缓冲液，0.5μLXbaI和0.5μLSpeI。在37°C水浴中孵育2小时。 </li><li>用XbaI和SpeI对C-Brick标准载体进行消化:向微量离心管中加入34μL超纯水，10μL质粒（1μg），5μL10x缓冲液，0.5μLXbaI和0.5μLSpeI 。在37°C水浴中孵育2小时。 </li><li>进行1％琼脂糖凝胶电泳，并用凝胶清洗系统试剂盒纯化步骤2.3.4的片段和来自步骤2.3.5的线性化载体;遵循制造商的协议。 </li><li>将片段和C-Brick载体连接:将2μL来自步骤2.3.5的线性化载体（50ng）和6μL来自步骤2.3.4的片段（200ng）加入到1.5mL微量离心管中。广告d 1μL10×T4 DNA连接酶缓冲液和1μLT4 DNA连接酶。在22°C孵育2小时。立即使用样品或将其冷冻保存在-20°C。 </li></ol></li><li>加入10μL产品，步骤2.1.8,2.2.9,2.3.3或2.3.7进行大肠杆菌感受态细胞化学转化 （DH10B）。 </li><li>将几个克隆接种到5-mL液体Luria-Bertani（LB）管中，并在37℃下在振荡器（220rpm）上孵育过夜。 </li><li>使用质粒制备试剂盒提取质粒;遵循制造商的协议。 </li><li>通过Sanger测序鉴定正确的克隆11 。 </li></ol> 3. C砖装配（ 图2 ） <ol><li> 用Cpf1消化C-Brick载体 <ol><li>加入22.5μL不含RNase的水，4μL10x Cpf1缓冲液，10μL来自步骤2.6（1μg）的质粒，0.5μLRRI和1μl＃181;将Cpf1（5μM）的L加到1.5-mL微量离心管中。 注意:Cpf1可以在以前的研究中按照协议在商业上购买或纯化4 。 </li><li>为了将外源DNA片段插入到T1和T2位点，加入1μL的crRNA-T2（10μM），并在37℃的水浴中孵育30分钟。 将 ddl crRNA-T1（10μM）和Cpf1（5μM）另外30分钟。 注意:另外，为了将外源DNA片段插入T3和T4位点，加入1μL的crRNA-T3（10μM），并在37℃的水浴中孵育30分钟。再加入1μL的crRNA-T4（10μM）30分钟。 </li><li>加入1μL热敏碱性磷酸酶，并将管在37°C水浴中孵育另外1 h。 </li><li>进行琼脂糖凝胶电泳，并使用凝胶清理系统试剂盒纯化载体。立即使用样品或将其冷冻保存在-20°C。 </li></ol></LI&gt; <li> 用Cpf1消化DNA片段 <ol><li>向1.5 mL微量离心管中加入21.5μL无RNase的水，4μL10x Cpf1缓冲液，10μL质粒，步骤2.6（2μg），0.5μLRRI和2μLCpf1（5μM）。 </li><li>要将外来DNA片段插入T1和T2位点，加入1μL的crRNA-T1（10μM）和1μL的crRNA-T3（10μM），并在37℃的水浴中孵育2小时。 注意:或者，加入1μL的crRNA-T2（10μM）和1μL的crRNA-T4（10μM），并在37℃的水浴中孵育2小时。 </li><li>运行琼脂糖凝胶电泳，并使用凝胶清理系统套件纯化该片段。立即使用样品或将其冷冻保存在-20°C。 </li></ol></li><li> C砖组装和进一步验证 <ol><li>连接外来DNA片段和C-Brick载体:加入2μL线性化载体（50ng）和6μL来自步骤3.2.3的DNA片段（200ng）至1.5mL微量离心管。加入1μL10×T4 DNA连接酶缓冲液和1μLT4 DNA连接酶。在22°C孵育2小时。立即使用样品或将其冷冻保存在-20°C。 </li><li>加入10μL连接产物（步骤3.3.1）进行大肠杆菌 （DH10B）感受态细胞的化学转化。 </li><li>将几个克隆添加到5 mL液体LB管中，并在振荡器上以220 rpm和37°C孵育过夜。 </li><li>使用质粒制备试剂盒提取质粒;遵循制造商的协议。 </li><li>通过Sanger测序鉴定正确的克隆11 。 </li></ol></li><li>执行新一轮C-Brick装配。步骤3.3.4中的正确克隆可以用作新的C-Brick向量或DNA部分，用于进一步迭代C-砖装配，遵循步骤3.1-3.3中相同的协议。 注意:T2&#39;和T3&#39;它被设计为T2和T3站点的备份（ 图1a ）。对于从步骤2.3.7获得的质粒，只有T2&#39;和T3&#39;可用于C-Brick标准组装。 </li></ol>

<ol>
	<li>Canton, B., Labno, A., Endy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refinement+and+standardization+of+synthetic+biological+parts+and+devices.">Refinement and standardization of synthetic biological parts and devices.</a> Nat Biotechnol. 26 (7), 787-793 (2008).</li><li>Shetty, R. P., Endy, D., Knight, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+BioBrick+vectors+from+BioBrick+parts.">Engineering BioBrick vectors from BioBrick parts.</a> J Biol Eng. 2, (2008).</li><li>Knight, 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> Idempotent Vector Design for Standard Assembly of Biobricks. , 1-11 (2003).</li><li>Li, S. Y., Zhao, G. P., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-Brick:+A+New+Standard+for+Assembly+of+Biological+Parts+Using+Cpf1.">C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1.</a> ACS Synth Biol. 5 (12), 1383-1388 (2016).</li><li>Anderson, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BglBricks:+A+flexible+standard+for+biological+part+assembly.">BglBricks: A flexible standard for biological part assembly.</a> J Biol Eng. 4 (1), (2010).</li><li>Liu, J. K., Chen, W. H., Ren, S. X., Zhao, G. P., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iBrick:+a+new+standard+for+iterative+assembly+of+biological+parts+with+homing+endonucleases.">iBrick: a new standard for iterative assembly of biological parts with homing endonucleases.</a> PLoS One. 9 (10), e110852 (2014).</li><li>Hsu, P. D., Lander, E. S., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157 (6), 1262-1278 (2014).</li><li>Wright, A. V., Nunez, J. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+Applications+of+CRISPR+Systems:+Harnessing+Nature's+Toolbox+for+Genome+Engineering.">Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering.</a> Cell. 164 (1-2), 29-44 (2016).</li><li>Jinek, M., 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=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Zetsche, B., 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=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163 (3), 759-771 (2015).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proc Natl Acad Sci U S A. 74 (12), 5463-5467 (1977).</li><li>Engler, C., Kandzia, R., Marillonnet, 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+one+pot,+one+step,+precision+cloning+method.">A one pot, one step, precision cloning method.</a> PLoS One. 3 (11), e3647 (2008).</li><li>Gibson, D. G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Casini, A., Storch, M., Baldwin, G. S., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bricks+and+blueprints:+methods+and+standards+for+DNA+assembly.">Bricks and blueprints: methods and standards for DNA assembly.</a> Nat Rev Mol Cell Biol. 16 (9), 568-576 (2015).</li><li>Cameron, D. E., Bashor, C. J., Collins, 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=A+brief+history+of+synthetic+biology.">A brief history of synthetic biology.</a> Nat Rev Microbiol. 12 (5), 381-390 (2014).</li><li>Keasling, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology+for+synthetic+chemistry.">Synthetic biology for synthetic chemistry.</a> ACS Chem Biol. 3 (1), 64-76 (2008).</li></ol>

作者没有什么可以披露的。

该协议描述了DNA组装标准C-Brick的程序。该协议中最重要的一步是C-Brick标准向量的线性化;载体的不完全切割可能严重影响成功率。另外，虽然Cpf1主要以"18-23"切割模式切割目标DNA序列，但也检测到两个碱基附近的不准确的切割，这可能在DNA组装后引起少量突变。因此，需要Sanger测序来验证组装的构建体。 C-Brick组件的效率低于以前研究中已经描述的传统限制酶和连接方法4 。影响组装效率的主要原因可能是Cpf1的裂解特性不准确。在将来，对于标准a的改进对于切割精度可能是非常有用的目前正在探索。 C-Brick也与BglBrick和BioBrick标准部分兼容。例如，可以用BglII和BamHI切割BglBrick部分，然后插入C-Brick标准载体的BamHI位点，产生C-Brick部分。但是，应该验证BglBrick部件的插入方向，并在装配后存在"GGATCT"疤痕。当从BioBrick部分（ 即 XbaI和SpeI消化）获得C-Brick DNA部分时，T2和T3位点将被破坏，并且可以使用两个备用Cpf1靶序列（T2&#39;和T3&#39;）。 由于Cpf1切割需要对RNA酶极其敏感的sgRNA，因此所有的物质都应该不含RNA酶。目前，所有C-Brick标准载体，无RNase的Cpf1核酸酶和crRNA均可在商业上订购12 。因此，C-Brick的程序实际上与Bio的简单砖标准。 在过去几年中，已经开发并广泛使用了几种有用的DNA组装方法，包括金门大会13 ，Gibson大会14等15 。然而，由于DNA装配标准有潜力提供具有成本效益，可靠性，高通量和自动装配反应5 ，标准可以促进新设计或重新设计的基因，遗传回路和途径的构建和测试15 ， 16，17 ，特别是在合成生物学领域。在目前公布的DNA装配标准中，C-Brick标准具有明显的优点和缺点4 （ 即连接效率）。如果提高结扎效率，今后可以广泛采用该标准。

DNA生物部件的标准化对合成生物学的发展至关重要1 。 DNA组装过程的发展可以取代特设实验设计，并消除在遗传组分装配到更大系统中时出现的许多意想不到的结果。 BioBrick标准（BBF RFC 10）是最早提出的DNA装配标准之一。它使用前缀序列（含有EcoRI和XbaI切割位点）和后缀序列（含SpeI和PstI切割位点） 2,3 。由于XbaI和SpeI具有互补性，所以用XbaI和SpeI切割的BioBrick DNA部件可以连接在一起，生成一个新的BioBrick，用于进一步的迭代装配。 已经使用BioBrick标准鉴定了一些缺陷4 。例如，它产生8-bp的瘢痕在DNA部分之间，其不允许构建融合蛋白。此外，上述四种类型的6-bp限制性位点必须从DNA部分中去除，这是非常不方便的。 BglBrick标准是为了解决第一个问题而建立的。它产生6-bp"GGATCT"瘢痕，产生Gly-Ser并允许多个蛋白质或蛋白质结构域的融合。 iBrick被开发来处理第二个问题6 。它使用识别长DNA序列的归巢内切核酸酶（HE）。由于HE识别位点在天然DNA序列中很少存在，所以iBrick标准可用于直接构建iBrick部分而不修改其DNA序列。然而，iBrick标准在DNA部分之间留下了21bp的瘢痕，这可能是其不受欢迎的原因。 近年来，群集定期交织的短回文重复序列（CRISPR）系统发展迅速7,8 。在CRISPR相关（Cas）蛋白中，来自化脓性链球菌的 Cas9核酸内切酶现在被广泛使用。它主要引入具有钝端的双链DNA断裂（DSB） 9 。 2015年，张和同事首次以Cpf1（来自Prevotella和Francisella 1的CRISPR）为特征。属于2类V型CRISPR-Cas系统，是CRISRP RNA（crRNA）引导型核酸内切酶10 。与Cas9不同，Cpf1引入了具有4或5 nt 5&#39;突出端的DSB 10 。基于此特征，Cpf1用于开发DNA组装标准C-Brick 4 。在C-Brick标准载体上，四个具有前缀T1 / T2和后缀T3 / T4的Cpf1靶位点位于生物部位的侧面;这与BioBrick标准相似。随着T2和T3位点的切割p导致互补的粘性末端，可以在部件之间产生"GGATCC"疤痕的同时执行DNA部件的迭代装配。值得注意的是，C-Brick标准有两个主要优点:识别长目标序列并留下短疤痕。由C-Brick产生的6bp"GGATCC"瘢痕编码Gly-Ser，其允许构建融合蛋白。此外，C-Brick标准也与BglBrick和BioBrick标准部分兼容。

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Comercial Oligonucleotide</td>
			<td style="width:160px;">Sangon Biotech</td>
			<td style="width:161px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">10x Taq PCR Buffer</td>
			<td style="width:160px;">Transgen</td>
			<td style="width:161px;">#J40928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Ultra Pure Distilled Water</td>
			<td style="width:160px;">Invitrogen</td>
			<td style="width:161px;">10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">5x RNA Transcription Buffer</td>
			<td style="width:160px;">Thermo Scientific</td>
			<td style="width:161px;">K0441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">T7 RNA polymerase</td>
			<td style="width:160px;">Thermo Scientific</td>
			<td style="width:161px;">#EP0111</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">NTP mixture</td>
			<td style="width:160px;">Sangon</td>
			<td style="width:161px;">#ND0056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">RRI(Recombinant RNase Inhibitor)</td>
			<td style="width:160px;">Takara</td>
			<td style="width:161px;">2313A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">RNA Clean &#38; Concentrator-5</td>
			<td style="width:160px;">Zymo Research</td>
			<td style="width:161px;">R1015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">UV-Vis Spectrometer</td>
			<td style="width:160px;">Thermo Scientific</td>
			<td style="width:161px;">Nano-Drop 2000c</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">2x Phanta Max Buffer</td>
			<td style="width:160px;">Vazyme</td>
			<td style="width:161px;">PB505</td>
			<td>PCR buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">dNTPs</td>
			<td style="width:160px;">Transgen</td>
			<td style="width:161px;">AD101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:316px;">Phanta Max Super-Fidelity DNA Polymerase</td>
			<td style="width:160px;">Vazyme</td>
			<td style="width:161px;">P505-d1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Ezmax for One-step Cloning</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">24303-1</td>
			<td>seamless assembly kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">5x Buffer for Ezmax One-step Cloning</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">32006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">BamHI</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#R0136L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">BamHI-HF</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#R3136L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">BglII&#160;</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#R0144L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">XbaI</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#R0145L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">SpeI</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#R3133L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">10x Buffer 3</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#B7003S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">10x CutSmart Buffer</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">B7204S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">10x T4 DNA ligase Buffer</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">32002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">T4 PNK</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">32206</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">T4 DNA ligase</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">32210</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">DpnI</td>
			<td style="width:160px;">NEB</td>
			<td style="width:161px;">#R01762</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">SV Gel and PCR clean-up system</td>
			<td style="width:160px;">Promega</td>
			<td style="width:161px;">A9282</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Plasmid Mini Kit I</td>
			<td style="width:160px;">Omega</td>
			<td style="width:161px;">D6943-02</td>
			<td>plasmid preparation kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">thermosensitive alkaline phosphatase&#160;</td>
			<td style="width:160px;">Thermo Scientific</td>
			<td style="width:161px;">#EF0651</td>
			<td>FastAP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">10x Cpf1 buffer</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">32008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Cpf1</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">32105</td>
			<td>FnCpf1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">thermocycler</td>
			<td style="width:160px;">Applied Biosystems</td>
			<td style="width:161px;">veriti 96 well</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">C-Brick standard vector</td>
			<td style="width:160px;">Tolobio</td>
			<td style="width:161px;">98101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">E. coli [DH10B]</td>
			<td style="width:160px;">Invitrogen</td>
			<td style="width:161px;">18297010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Luria-Bertani media (tryptone)</td>
			<td style="width:160px;">Oxoid</td>
			<td style="width:161px;">LP0042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Luria-Bertani media (yeast extract)</td>
			<td style="width:160px;">Oxoid</td>
			<td style="width:161px;">LP0021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:316px;">Luria-Bertani media (NaCl)</td>
			<td style="width:160px;">Sangon Biotech</td>
			<td style="width:161px;">B126BA0007</td>
			<td></td>
		</tr>
	</tbody>
</table>

protocols for c-brick dna standard assembly using cpf1

CRISPR相关蛋白Cpf1在CRISPR RNA（crRNA）的指导下切割双链DNA，产生粘性末端。由于这个特点，Cpf1已被用于建立称为C-Brick的DNA装配标准，其具有长识别位点和短瘢痕的优点。在标准C-Brick载体上，有四个Cpf1识别位点 - 前缀（T1和T2位点）和后缀（T3和T4位点） - 侧翼生物DNA部分。 T2和T3位点的切割产生互补的粘性末端，这允许用T2和T3位点组装DNA部分。同时，组装后部件之间产生短暂的"GGATCC"疤痕。由于新形成的质粒再次含有四个Cpf1切割位点，所以该方法允许DNA部分的迭代装配，这与BioBrick和BglBrick标准相似。概述使用C-Brick标准来组装DNA部分的程序在这里描述。 C-Brick标准可以被科学家，研究生和本科生甚至业余爱好者广泛使用。

<html>该协议证明了三种生色蛋白盒（cjBlue（BBa_K592011），eforRed（BBa_K592012）和amilGFP（BBa_K592010））的组装。首先，将上述三种基因和终止子的编码序列分别克隆到C-Brick标准载体中。将短DNA部分，启动子和终止子通过PCR扩增引入到C-Brick载体中，使用在5&#39;末端含有短DNA部分的引物。之后是自连接方法。首先从头合成三种染色体编码序列，然后通过无缝组装克隆到C-Brick标准载体中。在以前的研究中描述了引物4 。 之后，将cjBlue，eforRed和amilGFP序列与终止子和启动子序列以相同的程序连接"xfig"&gt;图2。将正确的构建体转化到大肠杆菌中 ，显示出美丽的颜色（ 图3a ）。随后，使用C-Brick标准进一步组装两个色表现盒，以产生更多的颜色（ 即，来自amilGFP加eforRed的红色，来自amilGFP加cjBlue的绿色，以及来自eforRed plus cjBlue的浅紫色; 图3b ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55775/55775fig1.jpg" /> 图1:C-Brick标准接口DNA序列和C-Brick标准载体。 （ a ）C-砖标准界面-T1，T2，T3，T4，T2&#39;，T3&#39;的序列可以被其对应的crRNA识别，然后使用Cpf1引入5&#39;突出端。 T2和T3（或T2&#39;和T3&#39;）位点的切割产生互补的挂起。设计了八个限制站点，C-Brick标准与BioBrick和BglBrick部分兼容。 （ b ）具有限制性消化位点的C-Brick标准载体的质粒图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55775/55775fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55775/55775fig2.jpg" /> 图2:C-Brick标准中DNA装配的工作流程。 Cpf1消化的T2和T3靶位点分别产生"GGATC"和"GATCC"的互补的粘性末端，其可以连接以产生短的"GGATCC"瘢痕。详细的程序可以在文本（"C-Brick assembly"）的步骤3中找到。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55775/55775fig2large.jpg" target="_blank">请点击这里</a><html>查看此图的较大版本。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55775/55775fig3.jpg" /> 图3:由C-砖标准组装的大肠杆菌加工结构产生的多彩细菌颜料。 （ a ）在板上生长的大肠杆菌中表达三种色素蛋白 。没有染色体的细菌（阴性对照）显示在第4 个平板上。 （ b ）在液体LB培养基中生长的大肠杆菌中表达三种色素蛋白和三种组装的双重色蛋白 。从1到6，结构表达eforRed（1），amilGFP（2），cjBlue（3），amilGFP加eforRed（4），amilGFP加cjBlue（5）和eforRed加cjBlue（6）。在单个板或单个微量离心管中的彩色细菌从单个序列验证的克隆培养。 les / ftp_upload / 55775 / 55775fig3large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 名称 </td><td colspan="3"> 序列 </td></tr><tr><td> T7-F </td><td colspan="3"> GAAATTAATACGACTCACTATAGGG </td></tr><tr><td> T7-T1-R </td><td colspan="3"> gaattcagtagaaagttgcgataaATCTACAACAGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC </td></tr><tr><td> T7-T2&#39;-R </td><td colspan="3"> ctagagtaaagcttgaattcagtaATCTACAACAGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC </td></tr><tr><td> T7-T2-R </td><td colspan="3"> ggatcctttctcctctttctagagATCTACAACAGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC </td></tr><tr><td> T7-T3-R </td><td colspan="3"> ggatccactagtctctagctcgaATCTACAACAGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC </td></tr><tr><td> T7-T3&#39;-R </td><td colspan="3"> ctctagctcgagaaattcaaggaATCTACAACAGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC </td></tr><tr><td> T7-T4-R </td><td colspan="3"> ttcaagggaaactgcagctagctATCTACAACAGTAGAAATTCCCTATAGTGAGTCGTATTAATTTC </td></tr></tbody></table> 表1:本研究中使用的crRNA转录模板的制备的寡核苷酸。 T7-F是顶链寡核苷酸，其他是底链寡核苷酸。序列中的小写字母将被转录成crRNA的引导序列。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 名 </td><td colspan="3"> RNA序列（5&#39;-3&#39;） </td></tr><tr><td> crRNA-T1 </td><td colspan="3"> GGGAAUUUCUACUGUUGUAGAUUUAUCGCAACUUUCUACUGAAUUC </td></tr><tr><td> crRNA-T2 </td><td colspan="3"> GGGAAUUUCUACUGUUGUAGAUCUCUAGAAAGAGGAGAAAGGAUCC </td></tr><tr><td> crRNA-T3 </td><td colspan="3"> GGGAAUUUCUACUGUUGUAGAUUCGAGCUAGAGACUAGUGGAUCC </td></tr><tr><td> crRNA-T4 </td><td colspan="3"> GGGAAUUUCUACUGUUGUAGAUAGCUAGCUGCAGUUUCUCCUUGAA </td></tr><tr><td> crRNA-T2&#39; </td><td colspan="3"> GGGAAUUUCUACUGUUGUAGAUUACUGAAUUCAAGCUUUACUCUAG </td></tr><tr><td> crRNA-T3&#39; </td><td colspan="3"> GGGAAUUUCUACUGUUGUAGAUUCCUUGAAUUUCUCGAGCUAGAG </td></tr></tbody></table> 表2:本研究中使用的crRNA序列。文中通过步骤1中的体外转录产生了crRNA（"crRNA的制备"）。

Watch this Scientific Journal Video about Protocols for C-Brick DNA Standard Assembly Using Cpf1 at JoVE.com

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR相关蛋白Cpf1可以由专门设计的CRISPR RNA（crRNA）引导，以在所需位点切割双链DNA，产生粘性末端。基于此特点，建立了DNA组装标准（C-Brick），并详细介绍了其使用方法。

使用Cpf1的C-Brick DNA标准装配的方案

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

protocols-for-c-brick-dna-standard-assembly-using-cpf1

Nanyang Technological University

Research

JoVE Journal

Genetics

8.2K 次观看.  Chinese Academy of Sciences.  CRISPR相关蛋白Cpf1可以由专门设计的CRISPR RNA（crRNA）引导，以在所需位点切割双链DNA，产生粘性末端。基于此特点，建立了DNA组装标准（C-Brick），并详细介绍了其使用方法。 

视频: Protocols for C-Brick DNA Standard Assembly Using Cpf1

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

字符串装配 grna 克隆 (stacr) 的可自定义协议

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

心脏细胞治疗中干细胞 dna 完整性的评估

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...