Name: rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis
Uploaded: 2014-02-05T17:30:00
Description: Watch this Scientific Journal Video about Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis at JoVE.com

我们感谢布拉施克，格伦​​沃尔德和WITTWER实验室的咨询和技术援助的成员。这项工作是由PCMC基金会，美国国立卫生研究院R01 MH092256和DP2 MH100008和优生优育基金会的研究经费＃1-FY13-425的3月，为广东健力宝的支持。

1。 DNA的制备<ol><li>制备的DNA的裂解缓冲液：50mM的KCl，10mM的Tris-盐酸的pH 8.3，0.3％吐温20，0.3％NP40。加入新鲜的蛋白酶K至对使用12的天1毫克/毫升的最终浓度。 </li><li>组织收集： <ol><li>对于成年鱼翅夹： <ol><li>麻醉鱼：将鱼在0.004％的MS-222（三卡因）解决方案。等到鳃运动减慢。 </li><li>把鱼放在一摞5-10的Kimwipes切一小片尾翼，约2-3毫米，用无菌刀片的。 </li><li>迅速将鱼与淡水回收标记?...

<ol>
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G., Ahmed, J. N., Arkell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+melt+analysis+(HRMA);+a+viable+alternative+to+agarose+gel+electrophoresis+for+mouse+genotyping.">High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping.</a> PloS one. 7 (9), (2012).</li><li>Vorkas, P. A., Poumpouridou, N., Agelaki, S., Kroupis, C., Georgoulias, V., Lianidou, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIK3CA+hotspot+mutation+scanning+by+a+novel+and+highly+sensitive+high-resolution+small+amplicon+melting+analysis+method.">PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method.</a> J. Mol. Diagn. 12 (5), 697-704 (2010).</li><li>Parant, J. M., George, S. 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PCR结合HRMA是一种功​​能强大的技术，斑马鱼基因分型。这种方法的优点是它的速度，鲁棒性，以及灵敏度来检测偶数的点突变。整个协议，从翅片夹熔化曲线分析，可在少于八小时由个人执行的。此外，该技术是适合于高通量筛选;不需要使用溴化乙锭;和密封所有PCR和分析步骤，这有助于减少污染的问题。 这种技术的一个关键步骤是PCR扩增子和引物设计。典型的长度为PCR?...

斑马鱼（ 斑马鱼 ）是脊椎动物模型系统广泛用于开发和疾病模型的研究。最近，众多的转基因和基因突变技术已经开发了斑马鱼。快速的转基因技术，通常根据一个TOL2座子系统1中，已合并为多个DNA片段组件2改进的克隆选择。锌指核酸酶（ZFN）和转录活化因子样效应物核酸酶（TALENS）已被用于靶向基因座在两个体细胞和生殖系细胞，斑马鱼3,4。这些技术可以有效地产生转基因动物，具有高频突变的创建和种系传递3,4。 尽管取得了这些进展，在传统的斑马鱼基因分型技术限制的诱变和转基因工具的全部功能。 PCR然后进行凝胶电泳，有时结合不受限制。Ñ​​酶消化，被广泛用于检测基因组的修饰，但很费时，并确定小插入或缺失不敏感。 TaqMan探针检测具有较高的初始成本，需要仔细优化。 PCR产物的测序可能需要几天时间，并且是不实用的大规模筛选。限制性片段长度多态性（RFLP）分析，只能判别影响的限制性内切酶识别位点的一个有限的范围内的SNP。 高分辨率熔解分析（HRMA），封闭管后的PCR分析方法，是一种最近开发的方法，该方法快速，灵敏，价格低廉，适合于筛选大量样品。 HRMA可用于检测单核苷酸多态性，突变，和转基因5-7。 HRMA是基于双链DNA热变性，并且每个PCR扩增子具有唯一的解离（熔体）特性5。样品可以分辨由于吨ø其不同的核苷酸组成，GC含量，或长度，通常在用荧光染料的组合，只有结合双链DNA 8。因此，HRMA可以基于不同的熔融曲线的特征区分不同基因型。因为HRMA使用廉价的试剂和是一个单步PCR后的过程中，它可用于高通量的策略。 HRMA是破坏性的，所以下面的分析将PCR扩增子可以用于其他应用。 HRMA已在许多生物和系统，包括细胞系，小鼠和人类9-11中得到应用。它的使用最近在斑马鱼被描述检测由锌指核酸酶（ZFN）和塔伦斯6,12,13突变。 在本文中，我们将描述如何在胚胎和成年斑马鱼进行基于PCR的HRMA（ 图1）。这个协议是适合于检测单核苷酸多态性，转基因，和基因突变，包括SIngle碱基对的改变，插入或缺失。

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		<tr height="42">
			<td height="42" style="height:42px;width:232px;">100 Reaction LightScanner Master Mix</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">HRLS-ASY-0002</td>
			<td style="width:157px;">www.biofiredx.com</td>
			<td style="width:167px;">Store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Hard-Shell PCR 96-well BLK/WHT Plates</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">HSP9665</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">MSB1001</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">96-Well LightScanner Instrument</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">LSCN-ASY-0040</td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Software with Call-IT 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:232px;">High-Resolution Melting Analysis 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Primer Design Software</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vector NTI Software</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:141px;"></td>
			<td>www.invitrogen.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine</td>
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			<td height="21" style="height:21px;width:232px;">Paraformaldehyde</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

斑马鱼是研究开发，造型疾病，并进行药物筛选功能强大的脊椎动物模型系统。最近的各种遗传工具的相继出台，其中包括诱导突变并产生转基因株系多种策略。然而，大规模的筛选是通过传统的基因分型方法，这是耗时耗力的限制。在这里，我们描述的技术来分析斑马鱼的基因型PCR结合高分辨率熔解分析（HRMA）。这种方法快速，灵敏，而且价格便宜，具有污染器物风险较低。基因分型的PCR与HRMA可以用于胚胎或成年鱼，包括高通量筛选的协议。

可以在一天内进行，或在步骤分离出的协议数天（工作的流程图示于图1）。 DNA提取之后，通过PCR扩增子的熔化和分析。的温度为扩增子的熔化取决于大小和GC含量，但一般就50˚C和95˚C端的温度是适当的（ 图2A和2B）。一旦熔体被执行时，荧光熔融曲线的分析通常需要不同的样本曲线的变化的归一化，使用前和后的熔融区域作为标准（ 图2C）。这?...

Watch this Scientific Journal Video about Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis at JoVE.com

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

PCR结合高分辨率的熔融分析（HRMA）被证明是一种快速，有效的方法，以斑马鱼的基因型。

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

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