We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).

注:按照有关放射性物质的安全使用和处置的所有机构的规定，包括但不限于个人防护装备的使用，如手套，防护眼镜，白大褂，和适当的丙烯酸盾牌。 注:标准缓冲液是由40毫米的HEPES-KOH，pH值7.5，50毫米钾谷氨酸，5毫摩尔DTT，0.1毫摩尔EDTA（以5倍浓度此缓冲区是预先定制的），每个的dNTP为0.3毫米的。 T7复制蛋白质如所述8，9，10纯化。含有单一的T7引发酶识别位点（5&#39;-GGGTC-3&#39;，5&#39;-GTGTC-3&#39;，5&#39;-TGGTC-3&#39;）的单链DNA可以从各种不同的DNA合成公司（长度可购买的单链DNA可取决于所选择的酶，T7引发酶，五聚体是最小的模板长度以合成全长的引物11，作为隐蔽C是必不可少的，但是，如果该引物是要通过聚合酶延伸，额外的核苷酸必须出现在模板的3&#39;末端。反应通过加入10mM的MgCl 2的最终浓度的启动，并在25℃温育。人工取样工作得很好，5秒以上的时间点。如果需要的时间点比短，建议使用一个快速骤冷流速仪以获得准确的数据。 1.多成交引物合成反应由手工取样<ol><li>在实验开始前，试样2甲酰胺染料缓冲液（93％（体积/体积）甲酰胺，50毫摩尔EDTA，0.01％二甲苯蓝和0.01％溴酚蓝）转换成8 1.5毫升聚丙烯管中。在一般情况下，管的数目等于所分析的时间点的数量。 </li><li>制备20微升混合物由1×缓冲液，0.1mM的ATP和CTP，0.25毫居里/毫升[α-32 P] CTP，0.1μMSSDNA模板和0.1μMGP4（表示为六聚体浓度 - 0.6μM单体）。 </li><li>在室温下孵育该混合物5分钟。 </li><li>除去使用移液器混合物2微升并在步骤1.1为无反应控制（时间零）制备的1.5毫升管2微升甲酰胺染料缓冲器混合。 </li><li>加入2微升的0.1M的MgCl 2反应混合物（以10mM的最终浓度）。 </li><li>在10秒的间隔手动收回使用移液器反应混合物2μL样品，并在步骤1.1编写的1.5 mL管predispensed甲酰胺染料混合。 </li><li>在95℃下热样品在热块5分钟。 </li><li>加载样品的等分试样到变性凝胶。由T7启动引发酶合成的小tetraribonucleotide底漆产品上的0.4毫米解析以及厚25％的丙烯酰胺:双丙烯酰胺（19:1），3M尿素，1×TBE（100毫摩尔Tris-硼酸盐，1mM EDTA）中。 </li><li>放在整个chromatogra凝胶PHY纸，用保鲜膜覆盖。干凝胶使用凝胶干燥。 </li><li>制备系列稀释的反应混合物的剩余部分，并当场加载的凝胶上色谱纸在相同体积， 即 ，发现稀释液4微升如果样品的4微升装载。 注意:这将作为标准曲线来确定在反应过程中形成的产物的浓度。例如，使用的TE缓冲液（10mM的Tris-HCl，pH值8.0，1mM EDTA）中很好地工作，跨越一系列五倍稀释1:25到1:15625（5 2 -5 6），但稀释应根据活动的水平进行调整。 </li><li>揭露使用磷光屏干胶和标准。量化在稀释系列中的放射性和描述8构建的标准曲线，12 注:T7 DNA聚合酶的夹杂物向反应混合物允许一个EXA矿井tetraribonucleotide引物，通过这种酶的使用量。最佳地，聚合酶浓度应饱和简化数据的解释。 0.4微米或更高的聚合酶浓度给出了良好的效果。此外，可以以这种方式被检验其他蛋白质，如单链DNA结合蛋白，在任引物合成或滞后链引发的效果。 </li></ol> 2.多成交引物合成反应中，用快速淬火仪<ol><li> 反应混合物的制备 <ol><li>制备溶液A的400微升，含有1x缓冲液，0.2μM的GP4六聚体，0.2微米的ssDNA模板，0.6毫的dNTPs，0.2mM的ATP和CTP（含0.5毫居里/毫升[α-32 P] CTP。 </li><li>制备400μL的溶液B，含有1x缓冲液和20mM的MgCl 2。 </li></ol></li><li> 快速淬火流量仪表的操作 <ol><li>开启说唱ID-淬火流量仪表;出现主菜单之后，该仪器的键盘上键入7到注射器驱动器移动到原位置。 </li><li>打开缓冲注射器位置"LOAD"。 </li></ol></li><li> 装载缓冲注射器 <ol><li>附上含有缓冲驱动注射器端口10毫升的注射器。更改注射器旋钮位置"LOAD"。 </li><li>使用10毫升注射器，填仪缓冲液储A和B用10mM的Tris，pH值7.5，1mM EDTA和填充骤冷溶液（250毫摩尔EDTA和0.2％SDS）中注射器℃。通过在进出推动柱塞除去气泡。 </li><li>更改旋钮的位置，以"火"。 2型调整注射器驱动器杆的位置。按"向下调整"按钮，直到缓冲区出来的退出循环，以降低注射器（连续7-下降; 8大降压; 9小步向下）。键入"ESC"键，返回到主菜单。 </li></ol></li><li> 清洗油管 <ol><li> ATTACH出口管线至真空。更改样本旋钮"刷新"。由旋钮设定到水平位置附近的缓冲注射器。打开真空和浸2冲洗循环的H 2 O代表10秒，然后在甲醇浸10秒。干循环通过吮吸〜15秒的空气，或直至干燥。 </li></ol></li><li> 装载样品 <ol><li>更改样本旋钮位置"LOAD"。将溶液A到1毫升注射器。注射器插入到样品负荷的港口之一。重复溶液B，并在相反的样品装载口放置。 </li></ol></li><li> 的时间点采集 <ol><li>在主菜单中，对于淬火流运行1型。输入反应时间（s），然后按"ENTER"。反应循环使用数量将被显示。切换到所需的反应循环。在步骤2.2.4重复清洗过程。 </li><li>打开样品旋钮"LOAD"。负载反应样品混合循环直到刚刚中央搅拌室外面。将所有旋钮"FIRE"，确认所有的旋钮都在正确的位置。 </li><li>将1.5毫升管到出口环结束。按下"GO"，以运行反应。在步骤2.2.4重复清洗过程。刷新缓冲区注射器A和B，直到他们击中了司机。 </li><li>重复步骤2.2.6.2-2.2.6.3为每个所需时间点。例外:请勿将包含启动试剂（ 如 ， 氯化镁在这种情况下）淬火零时间点控制时的解决方案。 </li><li>当收集完样本，输入"ESC"键返回主菜单。按"7"到缓冲针筒驱动器返回到其初始位置。 </li></ol></li><li> 仪器清理 <ol><li>除去反应混合物注射器。打开样品装载旋钮"LOAD"位置。在真空下，洗净用10ml 0.2N的氢氧化钠每个样品环，接着用10毫升0.3的NH 3 PO 4和10ml MeOH中。 </li><li>打开注射器旋钮"LOAD"位置。向下按所有3个SYR英格柱塞检索反应和淬火缓冲区。洗涤用5毫升水2 O注射器至少两次。 </li><li>用5毫升水2 O再次填充注射器和转旋钮"火"的位置。改变旋钮位置并打开真空，除去H 2 O.作为冲洗步骤2.2.4。关闭仪器。 按照步骤1.7-1.12描述分析产品:注意 。此外，T7 DNA聚合酶和其它复制蛋白可以在反应混合物中加入以分析其对引物合成或扩展的效果。 </li></ol></li></ol> 3.单成交引物合成反应 注:单成交引物合成/扩展试验也同样进行了多次失误的反应，有几个主要区别:这些试验遵循放射性标记的ATP到底漆或延伸产品的转化，基板但是浓度和蛋白质considera布莱高。另外，为了达到毫秒的分辨率，以使引物合成反应的分析，必须利用一个快速淬火流的机器。 <ol><li> 反应混合物的制备。 <ol><li>准备溶液A 400微升，含有1x缓冲，3μMGP4六聚体，6μM单链DNA模板，0.6毫米的dNTP，1毫米CTP和2纳米[γ-32 P] ATP。制备400μL的溶液B，含有1x缓冲液和20mM的MgCl 2。 </li></ol></li><li> 快速淬火操作流程 <ol><li>进行反应，如步骤2.2所述。如果需要的话，添加T7 DNA聚合酶或T7单链DNA结合蛋白在15μM和20μM的浓度，分别。在这种情况下，延伸产物积聚在允许用于反应的手动采样时间制度。 </li></ol></li></ol> 4.数据分析<ol><li>配合产品进度曲线非线性least-使用市售软件能够最小二乘的配合和/或数值积分平方模型。操作的细节将随所使用的软件变化，但以下是用于拟合数据的方程的广义形式。 </li><li>通过拟合进步曲线的线性方程确定的产物形成多个周转引物合成反应的速率。如果有显著曲率，通过将数据拟合到单指数函数使用初始速率方法13，14:Y = A（五- K t），其中y是产物浓度，A是反应振幅，k被所观察到的速率常数，t是时间，单位为秒。在时间= 0的切线的斜率是初始速率。 注:飞度前稳态引物合成反应的线性方程或预稳态突发方程Y = A（E - K BURST T）+率SS t，其中A是反应幅度，K 爆是预稳态突发观察到的速率常数，速率ss是稳态率SS = K 猫 [酶]），t为时间（秒）。适合单成交引物合成反应发展到一个单一的指数函数y = A曲线（E - K T）。在GP4的情况下，通过数值积分15拟合数据，以具有三个NTP缩合步骤，其中每个中间是在与酶平衡模型，使用市售的软件。 </li></ol>

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The authors have nothing to disclose.

在执行这些实验的最重要的因素是高活性的纯化的酶的可用性。在我们与GP4的工作，例如，我们发现，在缓冲液中纯化的酶的含50％甘油的存储（20mM磷酸钾，pH 7.4，0.1毫摩尔EDTA，1毫摩尔DTT）在-20℃导致减少制备过几个月的比活性。因此，我们现在在25mM的Tris-HCl，pH值7.5，50 mM氯化钠，0.1毫摩尔EDTA，1毫TCEP纯化GP4，和10％甘油的闪冻小等份用液氮并将其存储在-80℃。在使用快速淬火流仪，特别是采?...

DNA的复制是所有细胞生命的一个保守的特征。而复制组件的特性和数量有很大的不同，一般机制跨越进化1共享。这里，我们描述了基于凝胶的，不连续的动力学实验，使用的放射性底物，旨在通过T7启动复制体的组分理解体外滞后链DNA合成的动力学。 T7噬菌体的复制机制是很简单的，仅由四种蛋白质（DNA聚合酶，GP5，其持续合成因子， 大肠杆菌硫氧还蛋白，双官能引发酶-解旋GP4，和单链DNA结合蛋白，GP2的。 5）2。这一特性使得它来研究参与DNA复制保守的生化机制有吸引力的模型系统。 特别强调的是由T7 DNA形成的核糖核苷酸引物引发酶，一个critica升步骤中的DNA合成的起始。此外，还可以通过将其包括在反应混合物中检查这些引物由T7 DNA聚合酶或其他复制蛋白的用法。 T7启动引发酶 - 解旋酶，GP4，催化的引物DNA的合成在称为引发酶识别位点，或者PRS特定的DNA序列的形成，（5&#39;-GTC-3&#39;）。在PRS胞嘧啶是隐蔽，它是用于识别站点的必需的，但它不被复制到产品3。在引物合成的第一步由GP4涉及二核苷酸pppAC，然后扩展到三聚体，并最终官能四核苷酸引物pppACCC，pppACCA或pppACAC取决于序列在模板4的形成。这些引物然后可以通过T7 DNA聚合酶被用于启动DNA合成，这也是一个GP4辅助过程5,6。在这方面，引发酶域稳定极短的tetraribonucleotides与模板，防止其分解，以有利于保护底漆/模板到聚合酶活性位点7的方式从事DNA聚合酶。这些步骤（RNA引物的合成，引物越区切换到聚合酶和扩展）中重复多次循环复制后随链，必须与前导链的复制进行协调。 此处所描述的测定法是高度敏感的，并可以在一个温和的时间框架来执行。然而，它们是相对低的通量和必须十分小心以放射性物质的使用和处置行使。取决于在该反应进行时，人们可能会采用一种快速淬火仪在时间尺度适合用于在任一稳定或预稳态反应时间课程有意义的分析获得的样品的速度。最近，我们这里使用的描述测定提供我国证据的引物释放的从GP4的冈崎片段的开始时的引发酶域的重要性即此外，我们发现了为T7启动单链DNA结合蛋白，gp2.5调节作用的证据，在促进高效引物形成和利用8。

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			Tris pre-set crystals 
			pH 7.5</td>
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			<td height="63" style="height:63px;width:216px;">Magnesium chloride hexahydrate</td>
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			<td height="21" style="height:21px;width:216px;">1,4-Dithiothreitol</td>
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			<td height="63" style="height:63px;width:216px;">Sodium Dodecyl Sulfate</td>
			<td style="width:83px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">811030</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td style="width:119px;">4931-04</td>
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			<td style="width:83px;">PerkinElmer</td>
			<td style="width:119px;">BLU508H</td>
			<td>radioactive, take protective measures</td>
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			<td style="width:83px;">PerkinElmer</td>
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			<td>radioactive, take protective measures</td>
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			<td height="42" style="height:42px;width:216px;">100 mM dATP, dCTP, dGTP, dTTP&#160;</td>
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			<td align="right" style="width:119px;">77100</td>
			<td>four dNTPs part of set</td>
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			<td height="21" style="height:21px;width:216px;">100 mM ATP and CTP</td>
			<td style="width:83px;">Epicentre</td>
			<td style="width:119px;">RN02825</td>
			<td>part of NTP set</td>
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			<td height="84" style="height:84px;width:216px;">ssDNA constructs</td>
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			<td style="width:119px;">N/A</td>
			<td>custom sequences&#160;</td>
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			<td height="63" style="height:63px;width:216px;">methanol&#160;</td>
			<td style="width:83px;">Macron Fine Chemicals</td>
			<td style="width:119px;">3017-08</td>
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			<td height="42" style="height:42px;width:216px;">formamide</td>
			<td style="width:83px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">17899</td>
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			<td height="21" style="height:21px;width:216px;">bromophenol blue</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">B8026&#160;</td>
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			<td height="21" style="height:21px;width:216px;">cylene xyanol&#160;</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">X4126&#160;</td>
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			<td height="21" style="height:21px;width:216px;">acrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">A9099</td>
			<td>Toxic</td>
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			<td height="21" style="height:21px;width:216px;">N,N&#8242;-Methylenebisacrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">M7279&#160;</td>
			<td>Toxic</td>
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			<td height="63" style="height:63px;width:216px;">Urea</td>
			<td style="width:83px;">Boston Bioproducts</td>
			<td style="width:119px;">P-940</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Boric acid</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td align="right" style="width:119px;">2549</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rapid Quench-Flow Instrument&#160;</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">RQF-3&#160;</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kintek Explorer</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">N/A</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kaleidagraph&#160;</td>
			<td style="width:83px;">Synergy Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
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</table>

kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

在协议中的步骤1， 即所述，通过手动采样多个周转条件下，通过GP4催化的引物合成反应获得图1A所示的结果。这里，凝胶电泳后的范围内的产品可以通过使用在引物的合成反应（ 图1A）的α位与32 P标记的CTP进行观察。标记的前体，CTP，显示出最高的流动性和向凝胶的底部，接着通过GP4合成对应于二，三较慢迁移的物种?...

Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

DNA Replication

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7.3K 次观看.  Harvard Medical School. We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7. 

视频: Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

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遗传学

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

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 ...

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 ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

高密度DNA和RNA微阵列.光刻合成、杂交和制备大型核酸库

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...