Name: a fast silver staining protocol enabling simple and efficient detection of ssr markers using a non-denaturing polyacrylamide gel
Uploaded: 2018-04-20T13:30:00
Description: Watch this Scientific Journal Video about A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel at JoVE.com

这项工作由中国广东自然科学基金 (2015A030313500)、省级重点国际合作研究平台和广东省高等教育重点科研项目 (2015KGJHZ015)、科学与广东省烟草专卖管理技术方案 (201403、201705)、广东省科技计划 (2016B020201001)、全国大学生创新培训项目 (201711078001)。在本出版物中提及商号或商业产品, 纯粹是为了提供具体信息, 并不意味着美国农业部的建议或认可。美国农业部是一个平等机会提供者和雇主。

1. SSR 标记 PCR 产物的制备
<ol><li>准备所有的化学试剂和 PCR 反应, 包括模板 DNA (30 ng/µL), 2× pcr 大师组合 (包含 2× pcr 缓冲器, 每 dNTP 的0.4 毫米, 3 毫米氯化镁2, 0.1 U/µL Taq 脱氧核糖核酸聚合酶和染料), 10 µM 每向前和反向引物、蒸馏或去离子水 (dH2O)。 注: 本研究使用的 SSR 标记为 PT51333 (正向底漆序列: 5 '-GCACCTTTGGTTATCCGACA-3 ' 和反向引物序列: 5 '-TGCTTTAAGTCATGTACCAAATTGA-3 ') 和 PT50903 (正向...

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C., Pareek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+efficient+way+to+detect+small+polymorphic+bands+in+plants.">Simple and efficient way to detect small polymorphic bands in plants.</a> Genome Data. 5, 218-222 (2015).</li><li>Liu, W., 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=Development+of+a+simple+and+effective+silver+staining+protocol+for+detection+of+DNA+fragments.">Development of a simple and effective silver staining protocol for detection of DNA fragments.</a> Electrophoresis. 38, 1175-1178 (2017).</li><li>Wang, D., Shi, J., Carlson, S. R., Cregan, P. B., Ward, R. W., Diers, B. 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浸渍后的凝胶洗涤是一个关键步骤。洗涤时间和水量不足, 可能导致板材和凝胶表面的浸渍液不完全去除, 造成暗背景。适当的开发时间是另一个关键步骤, 过度开发可能导致黑褐色背景与低对比度图像的 DNA 片段。此外, 浸渍步骤显著影响 DNA 片段的染色效率。虽然在浸渍溶液中延长浸渍时间超过5分钟或增加 AgNO3量, 但不会显著改善染色质量, 将浸渍时间减少到少于3分钟或减少 AgNO3...

PCR 标记技术的发展使植物遗传学和育种学的研究发生了革命性的改变1。简单序列重复 (SSR) 标记是最常用和最多才多艺的 DNA 标记之一。其广泛的基因组覆盖率, 丰度, 基因组特异性和重复性是 SSR 标记的优点, 除了它们的呈共显性遗传检测异型基因型2。一些研究已经使用 SSR 标记来调查遗传多样性, 跟踪祖先, 构建基因连锁图, 并映射经济重要性状的基因3,4。
SSR 标记的 PCR 产物通常用琼脂糖或聚丙烯酰胺凝胶电泳进行分离, 然后用银染色或紫外光溴溴化物染色。聚丙烯酰胺凝胶中 DNA 片段的银染色比其他染色方法更敏感5,6 , 并已广泛用于检测 dna 片段, 如 SSR 标记7。
与许多生物实验室技术一样, 聚丙烯酰胺凝胶的银染色已稳步改善, 因为它第一次被报告为片段可视化技术在 1979年8。该技术最初被修改为检测 DNA 片段的巴萨姆et 等。6在 1991年, 然后由桑吉内蒂和同事在1994年改进9 。在最近几十年中, 该方法已进一步优化6、7、9、10、11、12、13、14,15. 然而, 大多数这些更新版本的协议仍然有一些缺点, 如高技术需求和长时间的固定和安装6, 这限制了这些协议的应用7, 11。为了在生物研究中常规应用银染色, 迫切需要采用低成本、高效的 DNA 片段检测相结合的最优协议。
此外, 聚丙烯酰胺凝胶可分为变性和非变性聚丙烯酰胺凝胶, 并可用于检测 SSR 标记使用银染色法。其效果和分辨率并无显著差异, 但非变性聚丙烯酰胺凝胶更易于处理, 且耗时较短16。
在以前的研究15的基础上, 本研究的目的是详细描述一个优化的银染色协议, 以便在非变性聚丙烯酰胺凝胶中快速、简便、低成本地检测 SSR 标记。

<table>
	<tbody>
		<tr>
			<td>PCR master mix (Green Taq Mix)</td>
			<td>Vazyme Biotech Co. Ltd, China</td>
			<td>#P131-03</td>
			<td></td>
		</tr>
		<tr>
			<td>50-2000 bp DNA Ladder</td>
			<td>Bio-Rad, USA</td>
			<td>#170-8200</td>
			<td></td>
		</tr>
		<tr>
			<td>DL500 DNA marker</td>
			<td>Takara Bio Inc., Japan</td>
			<td>#3590A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#10043-35-3</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#6381-92-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#79-06-1</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#110-26-9</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine</td>
			<td>Sangon Biotech Shanghai, China</td>
			<td>#110-18-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Bind-silane</td>
			<td>Solarbio Beijing, China</td>
			<td>#B8150</td>
			<td></td>
		</tr>
		<tr>
			<td>AgNO3</td>
			<td>Sinopharm Chemical Reagent Beijing Co.,Ltd, China</td>
			<td>#7761-88-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Tianjin DaMao Chemical Reagent Factory, China</td>
			<td>#50-00-0</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2CO3</td>
			<td>Tianjin DaMao Chemical Reagent Factory, China</td>
			<td>#497-19-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>HNO3</td>
			<td>Guangzhou Chemical Reagent Factory, China</td>
			<td>#7697-37-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2S2O3.5H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co.,Ltd, China</td>
			<td>#10102-17-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Eriochrome black T(EBT)</td>
			<td>Tianjin DaMao Chemical Reagent Factory, China</td>
			<td>#1787-61-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tray</td>
			<td>Shanghai Yi Chen Plastic Co., Ltd, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>TS-1 Shaker</td>
			<td>Qilinbeter JiangSu, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>BenQ M800 Scanner</td>
			<td>BenQ, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DYY-6C Power supply</td>
			<td>Beijing Liuyi Instrument Factory, China</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>High throughout vertical gel systems, JY-SCZF</td>
			<td>Beijing Tunyi Electrophoresis Co., Ltd, China</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fast silver staining protocol enabling simple and efficient detection of ssr markers using a non-denaturing polyacrylamide gel

简单序列重复 (SSR) 是植物和动物遗传研究和分子育种项目中最有效的标记之一。银染色是一种广泛应用于聚丙烯酰胺凝胶中 SSR 标记检测的方法。然而, 传统的银染色协议在技术上要求和耗时。与许多其他生物实验室技术一样, 银染色协议已稳步优化, 以提高检测效率。在这里, 我们报告一个简化的银染色方法, 大大降低试剂成本, 提高检测分辨率和图像清晰度。新的方法需要两个主要步骤 (浸渍和开发) 和三试剂 (硝酸银, 氢氧化钠和甲醛), 和只有7分钟的加工为非变性聚丙烯酰胺凝胶。与以前报告的协议相比, 这种新方法更容易、更快, 并且使用较少的化学试剂进行 SSR 检测。因此, 这种简单、低成本、有效的银染色协议将通过快速生成 SSR 标记数据, 有利于遗传图谱和标记辅助育种。

采用相应的 SSR 引物对 amplicons 甘蓝和烟草进行了 PCR 制备。电泳后, 采用上述银染色协议对聚丙烯酰胺凝胶进行染色, 明确检测到 SSR 标记的带状模式 (图 1)。
为了比较不同银染色协议的检测效率, 采用聚丙烯酰胺凝胶电泳法分离了烟草和开花白菜中 SSR 标记的 PCR 产物, 并用五的银染色方法进行了可视化。?...

Watch this Scientific Journal Video about A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel at JoVE.com

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

在这里, 我们报告一个简单和低成本的银染色协议, 只需三试剂和7分钟的处理, 并适合快速生成高质量的 SSR 数据的遗传分析。

一种快速的银染色协议, 能够使用非变性聚丙烯酰胺凝胶对 SSR 标记进行简单而有效的检测

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

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