Ministry of Science and Technology of Taiwan provided financial support of this research under contracts MOST-103-2221-E-002 -097 -MY3.

1.检测方法MNP 注意:本节介绍了一种基于金纳米粒子的杂交介导的生长检测MNP的过程。 <ol><li>制备探针DNA（5&#39;-硫醇GAGCTGGTGGCGTAGGCAAG-3&#39;）的浓度为100μM的溶液。 </li><li>准备探针DNA修饰的金纳米粒子（金纳米粒子探针）的颗粒。21 注意:此处所用的量取决于探针DNA修饰金纳米粒子（金纳米粒子探针的）颗粒的要求。它因此是依赖于要进行的实验次数。我们通常超额准备金纳米粒子探测器粒子作为备用。在这些步骤中使用的体积是可调的和灵活的。 <ol><li>在浓度为1 OD /毫升（100μM，在步骤1.1中制备）添加探针DNA金纳米颗粒的溶液（13纳米，17纳米）。 </li><li>带来十二烷基硫酸钠的最终浓度（NAC 12 H 25 SO 4，SDS）和磷酸盐缓冲液（PBS）中，以0.01％和0.01男，respectivel年。 </li><li> 20分钟后，带来的氯化钠（NaCl）浓度为0.05M的用NaCl（2M）和PBS（0.01M），同时维持在0.01％SDS的溶液中，并孵育20分钟。 </li><li>增加的0.1M的增量NaCl浓度至1M在20分钟的时间间隔的终浓度。 </li><li>与在23℃下在涡旋混合器上振摇孵育过夜。摇动速度不只要DNA样品和金纳米粒子探针成为杂交影响实验。 </li><li>离心金纳米颗粒在7000×g离心30秒，并去除上清。 </li></ol></li><li>在25纳米（金纳米粒子探针溶液）的浓度添加金纳米粒子探针粒子到去离子水（DI水）。 </li><li>制备的靶DNA样品（完全互补:5&#39;-CTTGCCTACGCCACCAGCTC-3&#39;;三碱基对不匹配:5&#39;-CTTGCCTAC TTT ACCAGCTC-3&#39;; 6个碱基对的错配:5&#39;-CTTGCCT TTTTTT CCAGCTC-3&#39;）的浓度0.06，0.11，0.17，0.20，0.30，0.50微米，分别。 </li><li>添加金纳米粒子探针溶液（6微升，25纳米）到DNA样品（350微升）用NaCl（14μM）。 </li><li>摇DNA样品和金纳米粒子探针的混合物用涡旋混合器在23℃下5分钟进行DNA杂交。 </li><li>对于金纳米粒子的生长，添加NH 2 OH（6微升，400毫摩尔）和氯金酸4（6微升，25.4毫摩尔）该溶液中（DNA样品和金纳米粒子探针的混合物）。金纳米粒子的生长需要完成大约30秒。颜色的变化稳定保持1个多小时。 注意:用氯金酸皮肤接触可能会产生严重的毒性作用。使用前请咨询材料安全数据表（MSDS）。请戴手套，并使用通风橱使用氯金酸4时。 </li></ol> 2.气动操纵液滴平台的制造注:基于PDMS滴操控平台由两部分组成:一名PDMS膜（100微米）用超疏水性表面和空气沟道层（5毫米）。没有的MEMS工艺中，常见的加工过程被用于制造该装置，其中包括计算机数字控制（CNC）微机械加工制造的模具中，PDMS铸造和复制的微流体部件的快速原型设计，并激光微加工用于制造超疏水表面（见图3）。 <ol><li>使用CNC机床配备有钻头（0.5毫米）到产生PMMA基主模具（ 见图3）具有微结构。使用钻头7位毫米/秒的进给速度和旋转26000 rpm的速率。使用鼓风机和DI水以除去PMMA废料和清洁主模具的表面上。 </li><li>制备的PDMS基底和在比10的固化剂的混合物:1。脱气干燥器内的混合物30分钟，或直到所有的气泡都被删除。 </li><li>倒入PDMS进入次È母模，并在烘箱中烘烤3小时的PDMS在60℃，得到的气室的逆微结构（PDMS膜）和空气通道（PDMS层）。 </li><li>取下母模（用手）的PDMS层。冲PDMS层的空气吸入口上的孔。 </li><li>放PDMS膜，与PDMS层和玻璃基板到氧等离子体处理系统的室中。氧等离子体处理后，一起在电炉上（90℃）10分钟粘合PDMS膜，与PDMS层和玻璃基板。 </li><li>把复合芯片进入激光切割机（PDMS膜面朝上）。导入.dwg文件来定义超疏水区（15毫米×12毫米）。参见图3。直接与二氧化碳激光雕刻机的PDMS膜的PDMS膜（功率12 W，扫描速度106.68毫米/秒）创建超疏水区域。 </li><li>清洁用DI水的超疏水性表面洗远表面上的碳化残留物。 </li><li>通过电磁阀连接空气吸入口和真空泵（ 见图4）。电磁阀到计算机和控制连接与数字I / O USB模块。 </li></ol> 3.气动平台的MNP的检测操作注:这部分描述的操作以色度和迅速查明气动液滴操纵平台上的MNP（ 见图5）。所有步骤取在23℃（环境温度）的地方，相对湿度85％。 <ol><li>放置靶DNA样品的液滴（10微升，0.5μM）上的充气液滴操纵平台的超疏水性区域。 </li><li>放置金纳米粒子探针的液滴（10微升，25纳米）上的充气液滴操纵平台的超疏水性区域。 </li><li>控制吸气使PDMS膜的偏转用的电磁阀和一个SImple程序（ 例如，Labview的）。提供在PDMS膜使得液滴相互碰撞和聚结在沿液滴（含有靶DNA和金纳米粒子探针分别）的迁移路径各空气室的空气吸入。使用-80千帕（表压）的真空泵的工作压力。 <ol><li>可替代地，只要手动作为微滴充分混合控制空气吸入。用手，连接或断开真空泵和空气室的入口与在测试的早期阶段的管。 </li></ol></li><li>控制吸气滚动聚结的液滴前进和后退用电磁阀的控制系统，以提高混合效率为3分钟。靶DNA与金纳米粒子探针变得很好地混合，在3分钟内进行杂交。使用的驱动频率和5赫兹和-80千帕（表压）的工作压力，分别。 </li><li>放置装有NH 2 OH（2微升，400毫摩尔）和氯金酸的液滴4（2微升，25.4毫摩尔）上的充气液滴操纵平台的超疏水性区域。 </li><li>控制吸气（-80千帕表压），以让液滴彼此和聚结发生碰撞。则控制吸气（5赫兹和-80千帕表压），以让所述聚结的液滴滚向前和向后以提高混合效率，持续1分钟。 </li><li>测量和记录用肉眼聚结液滴的颜色。 </li></ol>

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The authors declare that they have no competing financial interests.

在这个协议中，一个简单的比色方法检测MNP可以在浓度范围为0.11-0.50微米的微离心管中实现。此外，所提出MNP检测方法上的充气液滴操作平台，该平台具有用于DNA筛查和其它生物医学应用的高电位下进行。在实践中，样品DNA浓度的可检测范围取决于的操作平台的混合效率。以确保该聚结的液滴充分混合，在该协议中的关键步骤是依赖于聚结液滴的大小吸气（压力和频率）的控制。完全互补的样本DNA和不匹配的样本DNA之间的杂交的亲和力的差异，很难用肉眼辨别为一个长的DNA序列片段。这种检测技术的局限性是，因此样品DNA的片段长度。对于长期DN样本一个序列，尺寸或生长金纳米粒子的构象类似于含有各种错配多种多样的DNA样品。 DNA样品与探针的错配出现在这个协议中的中间段。具有相同的长度和样本DNA的同聚不匹配，错配的位置也可能影响金纳米粒子的杂交亲和力和生长的大小。许多疾病，目前正在追查到特定的基因缺陷。对特定疾病的基因组数据库仍在扩大和提高。一旦特定的DNA片段的DNA序列变异被证实是负责特定疾病，专门设计的探针（具有适当的长度和该DNA的错配位置）可以被用于获取样本DNA的错配的数量的基础上可检测的浓度范围内探测在这项工作中已经测试。 建议的气动操纵液滴平台执行编在一个开放的环境。液滴的蒸发增加了DNA样品的浓度和影响比色读出的精度。以提高MNP分析的精度，控制环境温度和湿度是必需的。激光微加工施加在所提出的平台来制造超疏水性表面。在PDMS膜的超疏水下不熔滴操作（&gt; 20倍）的多次迭代坚持。超疏水性的​​损失减小这种液滴操纵平台的流动性和可操作性。一旦液滴操纵平台失去的表面上的超疏水性，remodifying的PDMS膜的超疏水性（重复步骤2.6-2.8）是必需的。更持久的超疏水表面的简单的制造正在将来审议。在这个协议中，我们使用一个电磁阀和一个简单的控制程序（与数据采集设备），以控制第Ë空气吸入。吸气的控制可以用其他方式或装置，包括但不限于使用电磁阀的使用。 在这个手稿，我们引入的DNA检测的简单比色方法和气动液滴操纵平台的结合。这两种技术是独一无二的，极具潜力;我们没有发现任何替代的DNA检测方法（也就是既快速和视觉）来实现相同的结果。在一个开放的表面，包括optowetting，22介，23电，24振动25和热水瓶毛细管启动26已报道的一些液滴操作技术。所有这些液滴操纵技术的缺点在我们以前的工作进行了讨论。20所提出的气动平台比上述方法更加生物相容的。 该技术检测的多核苷酸多态性Ø呐气动液滴操纵平台是在生物化学领域中，这意味着试样和试剂可直接装载和移液器收集研究者简单。所提出的平台的整合正与一个自动控制系统和用户接口的用于在未来改进的和广泛使用的设计进行的。

单核苷酸多态性（SNP），这是在DNA序列的单碱基对差异，是最常见的遗传变异之一。目前的研究报告，单核苷酸多态性与疾病风险，药效和通过影响基因功能的个人副作用有关。1,2-最近的研究还表明，双或多点突变（多核苷酸多态性）引起特定疾病和个体在疾病的影响差异。3,4核苷酸多态性的检测是因此病预检势在必行。为快速检测序列特异性寡核苷酸的简单而有效的方法，在过去的二十年是高度发达的1,5电流的方法来识别DNA突变通常涉及程序，包括探针固定，荧光标记，凝胶电泳等 ，6,7但这些方法一般需要很长的分析过程，expen西伯设备，训练有素的技术人员，以及样品和试剂的消耗显著。 与表面积与体积和独特的物理化学性质的大比率的纳米粒子是作为用于特定的生物标记物的高灵敏度和廉价的检测平台的理想材料。金纳米颗粒（金纳米粒子）被广泛用于DNA检测的，因为它们的大能力与寡核苷酸探针进行修改。8-10 SNP检测技术是使用金纳米粒子还开发11-13在这项工作中，我们采用了一种新颖的比色方法检测多核苷酸多态性（MNP）通过14金纳米粒子探针的DNA杂交介导的生长，这种简单的和快速的探测方法是基于这样的理论缀合到单链DNA（ssDNA）或双链DNA（dsDNA）的的不同长度金纳米粒子影响金纳米粒子的生长的大小和形状（参见图1）。15的DNA检测的这种方法设有试剂的消耗量小，小试验的持续时间（几分钟），并且没有热控制一个简单的过程，是前瞻性适用于临床诊断和国内医学筛查。 几个微流体系统以检测DNA序列已被开发; 16这些微流体系统中，从传统的实验方案演化而来，需要更少的块的大型设备，并简化了实验方案，以提高灵敏度，检测极限和特异性的DNA生物传感器。在微流体系统的DNA检测方法仍需要，但是，信号放大和荧光读取器的随后的处理的仪器如PCR（聚合酶链式反应）机对单个读出，以确定异构SNP。17,18开发一个简单的无随后的处理平台，以直接读出的多核苷酸多态性的结果是非常需要的。比起很好用的，传统的，封闭的微流体系统，开放式表面微流体装置有为地提供一些优势，比如一个明确的光路，一个简单的方法来访问样本，直接无障碍环境并没有容易形成气穴或界面阻塞通道19我们以前的工作引入开放表面的液滴操作（ 见图2）。20简单气动平台在此平台上，液滴可同时运输和使用抽吸力从驱动能量而不干扰操纵，其中有一个在生物和化学的应用潜力巨大。因此，该气动平台利用结合使用金纳米粒子探针的DNA杂交介导的生长的概念比色方法执行DNA样品的操纵对MNP的检测。 本文介绍的协议描述多核苷酸多态性的一个简单的目视检测气动操纵液滴平台上的开放面。这项工作证实，多核苷酸多态性是用肉眼可检测的;建议的气动平台，适用于生物和化学应用。

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">PDMS</td>
			<td style="width:204px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">benchtop engravers</td>
			<td style="width:204px;">&#160;Roland DG</td>
			<td style="width:119px;">EGX-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">laser cutting machine</td>
			<td style="width:204px;">Universal Laser Systems, Inc.</td>
			<td style="width:119px;">VLS 3.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Oxygen plasma treatment system</td>
			<td style="width:204px;">Femto Science Inc. Korea</td>
			<td style="width:119px;">CUTE-MPR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solenoid valve</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td>home built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">vacuum pump</td>
			<td>ULVAC KIKO, Inc.</td>
			<td style="width:119px;">DA-30D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">13-nm AuNP solution</td>
			<td style="width:204px;">TAN Bead Inc., Taiwan</td>
			<td style="width:119px;">NG-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNA (with 5 -end labeled thiol)</td>
			<td style="width:204px;">MDBio, Inc., Taiwan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">phosphate buffered saline (PBS)</td>
			<td>UniRegion&#160;Bio-Tech,. Taiwan</td>
			<td>PBS001-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:204px;">J. T. baker</td>
			<td style="width:119px;">4095-04</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Hydroxylamine solution (NH2OH)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:119px;">467804</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Chloroauric acid (HAuCl4)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>G4022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium chloride (NaCl)</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">vortex mixer</td>
			<td style="width:204px;">Digisystem Laboratory Instruments Inc.</td>
			<td style="width:119px;">VM-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">centrifuge</td>
			<td style="width:204px;">Hermle Labortechnik GmbH.</td>
			<td style="width:119px;">Z 216 MK</td>
			<td></td>
		</tr>
	</tbody>
</table>

the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform

是在一个开放的表面上液滴的气动操纵平台进行一个简单直观的方法来检测多核苷酸多态性（MNP）。这种方法比色检测DNA的基础上金纳米粒子探测器（探头金纳米粒子）的杂交介导的生长。该金纳米粒子的生长尺寸和结构通过与探针杂交的DNA样品的数量支配。根据纳米颗粒的具体大小 - 和形状依赖性的光学特性，错配的在样品DNA片段与探针的数目是能够被区分。试验是分别通过含有试剂和DNA样品的液滴中进行，被运送并与基于PDMS柔性超疏水性膜的受控气动吸入混合气的平台上。液滴可以同时并准确传递一个开放表面上所提出的气动平台，是没有副作用EFF高度的生物相容性液滴内的DNA样品的等。两个提出的方法相结合，该多核苷酸多态性可在视线气动液滴操纵平台上被检测;没有额外的仪器是必需的。从平台到最终结果上安装的液滴的步骤需要不到5分钟，比用现有方法少得多。此外，该组合MNP检测方法仅需要10微升，在每个操作中，这比一个宏系统的显着更小的样品体积。

在这项工作中，三种DNA样品使用检测的简单且新颖的方法通过金纳米粒子探针的DNA杂交介导的生长测试。探针DNA和三种DNA样本的序列，具体而言，CDNA（完全互补的探针DNA），TMDNA（三碱基对的错配的DNA），和SixMDNA（6个碱基对的错配的DNA）的协议步骤1中列出。此处所测试的DNA样品的探针的错配是无论在DNA样品的中间段。 图6示出了用于在不同的浓度的DNA样品的生长金纳米粒子溶液的颜色。对于完全匹配DNA（CDNA），解决方案的色调从粉红到靛蓝变化，然后以透明随着DNA浓度。为TMDNA，溶液的颜色从粉红到暗紫色变为再到靛蓝，和用于SixMDNA，颜色从粉红变为暗粉色随DNA浓度。单独的Ð<html> NA样本具有不同的杂交亲和力导致金纳米粒子的不同的增长规模和解决方案的多样色彩的探针DNA。的TMDNA样品，与从探针三碱基对的突变，具有比所述cDNA样品，这引起了金纳米粒子的较小的生长尺寸杂交亲和力更小。如在图6的结果显示，有cDNA和TMDNA样品之间的颜色的差别，特别是在一个大的DNA浓度。通过与探针许多错配，SixMDNA样品具有弱杂交亲和性探针，导致偶联至金纳米粒子几双链DNA，甚至与DNA浓度大于0.2微米以上。金纳米粒子的生长尺寸从而小此SixMDNA样品中，使金纳米粒子溶液从粉红色改变为紫红色，这是不很明显用肉眼测量。基于所述彩色图像中，cDNA，TMDNA和SixMDNA是从0.11的DNA浓度为0.50μM的大致区别。 _content"FO: - together.within页保持="1"&gt;使用这里提出的方案和方法，为开放式的表面微流体简单且新颖的基于液滴操纵平台证实使用气动吸力作为力来产生。可以不中断（ 见图7A）来实现到生物样品的DNA在此情况下，PDMS膜，液滴的轻便运输和操纵的表面变形，在液滴。MNP的新和视觉检测表现出对这个气动开放表面的液滴操作的平台。正如在图7B中示出的结果，DNA错配的检测是用肉眼很容易观察到，在原始状态下，金纳米粒子探针的液滴表现出红色。的最大光吸收金纳米粒子探针发生在520nm处，这归因于表面等离子体共振（SPR）。与各DNA样品的液滴，具有充分ç金纳米粒子的靶 - 探针DNA双链杂交后<html> omplementary DNA（CDNA）样品液滴变得透明，象增加的金纳米粒子的SPR功能消失。与此相反，金纳米粒子杂交的三个碱基对错配的DNA（TMDNA）和6个碱基对的错配的DNA（SixMDNA）分别为蓝色和紫色。用这种方法，检测DNA的所提出的平台和方法相结合，并以简单的方式来实现。 <img alt="图1" src="/files/ftp_upload/54424/54424fig1.jpg" /> 图1.从金纳米粒子硫醇修饰的ssDNA的杂交比色检测的MNP。硫醇改性的单链DNA（ssDNA）或双链DNA（dsDNA）的的原则的影响的金纳米粒子的生长的大小和形状，和金纳米粒子引起不同的光学特性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/54424/54424fig2.jpg" /> 图2.气动操纵液滴平台的示意图。有气动吸为力量，以使基于PDMS灵活的超疏水膜的偏转，在膜上水滴变得从而激活。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig2large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <img alt="图3" src="/files/ftp_upload/54424/54424fig3.jpg" /> 图3.气动操纵液滴平台的研制。建安包括计算机数控（CNC）微机械加工，铸造PDMS或复制技术和激光微加工。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig3large.jpg" target="_blank">请点击此处查看LARG</a>呃版本这个数字。 <img alt="图4" src="/files/ftp_upload/54424/54424fig4.jpg" /> 图4的充气微滴操纵平台的实验装置。真空泵产生的空气吸入是提供一种在空气室的减弱的压力和使PDMS膜液滴操作的偏转。真空泵，并通过电磁阀连接在空气通道的入口。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54424/54424fig5.jpg" /> 图5.本MNP检测技术的操作的示意图。含有靶DNA的液滴和金纳米粒子探针装在平台上，混合和HYBridized。含有NH 2 OH和氯金酸4液滴被装载在平台上，并混合金纳米粒子的生长。最后，金纳米粒子的目标探针DNA双链转移的最大吸收和改变了颜色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54424/54424fig6.jpg" /> 图6.变化与浓度的各种DNA样本的色彩。对于充分互补DNA（cDNA），溶液的色调从粉红色变化随DNA浓度成靛蓝。对于TMDNA，解决方案的颜色从粉红色变为暗紫色，并SixMDNA，颜色从粉红变为深粉红色随DNA浓度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig6large.jpg" target="_blank">请点击这里查看LAR</a>GER版本这个数字。 <img alt="图7" src="/files/ftp_upload/54424/54424fig7.jpg" /> 图7.气动平台， 一个 图像液滴 ），通过吸风; B控制）金纳米粒子的提议气动操纵液滴平台上的相应的目标探针DNA双螺旋的照片气动平台上液滴的运输序列图像。驱动频率和工作压力的操作条件分别为5赫兹和-80千帕（表压）。在原来的状态下，金纳米粒子探针的液滴表现出红色。在cDNA的存在金纳米粒子的靶 - 探针DNA双链是透明的。与TMDNA和SixMDNA杂交的金纳米粒子探针是蓝色和紫色分别。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。&lt;</a>/ A&gt;

Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

这项工作提出了一个简单直观的方法来检测气动操纵液滴平台的多核苷酸多态性。与所提出的方法中，在整个实验，包括液滴操作和多核苷酸多态性检测，可以接近23°C不先进仪器的帮助下进行。

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

the-visual-colorimetric-detection-multi-nucleotide-polymorphisms-on

Nanyang Technological University

Research

JoVE Journal

Genetics

7.5K 次观看.  National Taiwan University. 这项工作提出了一个简单直观的方法来检测气动操纵液滴平台的多核苷酸多态性。与所提出的方法中，在整个实验，包括液滴操作和多核苷酸多态性检测，可以接近23°C不先进仪器的帮助下进行。 

视频: The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

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 ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively and efficiently. Moreover, given the infrequency of these experiments it is imperative that their impacts be maximized. The rapid advancement of omics technologies offers an opportunity to dramatically increase the volume of data produced from precious spaceflight specimens. To capitalize on this, NASA has developed the GeneLab platform to provide unrestricted access to spaceflight omics data and encourage its widespread analysis. Rodents (both rats and mice) are common model organisms used by scientists to investigate space-related biological impacts. The enclosure that house rodents during spaceflight are called Rodent Habitats (formerly Animal Enclosure Modules), and are substantially different from standard vivarium cages in their dimensions, air flow, and access to water and food. In addition, due to environmental and atmospheric conditions on the International Space Station (ISS), animals are exposed to a higher CO2 concentration. We recently reported that mice in the Rodent Habitats experience large changes in their transcriptome irrespective of whether animals were on the ground or in space. Furthermore, these changes were consistent with a hypoxic response, potentially driven by higher CO2 concentrations. Here we describe how a typical rodent experiment is performed in space, how omics data from these experiments can be accessed through the GeneLab platform, and how to identify key factors in this data. Using this process, any individual can make critical discoveries that could change the design of future space missions and activities.

The NASA GeneLab platform provides unfettered access to precious omics data from biological spaceflight experiments. We describe how a typical mouse experiment is conducted in space and how data from such experiments can be accessed and analyzed.

利用开放接入美国宇航局基因实验室平台探索航天对小鼠生理的影响

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

墨西哥鱼基因功能的操纵

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...