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 (正向底漆序列: 5 '-AAATTTCTTTCGGTGTGATAACTG-3 ' 和反向底漆序列: 5 '-AGAGACTGCCGTTTGATTTGA-3 ') 为烟草和 CX-43 (向前底漆序列: 5 '-TGGGGATGTGAGCTTCTTCT-3 ' 和反向引物 sequence:5 '-AGGGTTCCTTTGGGGTGATA-3 ') 和 CX-157 (向前底漆序列: 5 '-TCGACGCTGACTTCACTGAC-3 "和反向引物序列: 5 '-GGACAGCTTCACACATTTGC-3 ') 为开花大白菜。</li>
	<li>通过添加2µL 模板 DNA、5µL 2× pcr 总混合、每向前底漆和反向底漆0.2 µL 和 dH2.6O 的2µL, 制备10µL 的 pcr 组合物进行扩增。</li>
	<li>执行 PCR 放大反应在一个 thermocycler 的初始步骤94°c 为5分钟, 跟随38周期四十五年代在94°c 为变性, 四十五年代在55°c 为底漆退火并且1分钟在72°c 为引伸, 并且最后的延长步骤 10 min 在72°C;然后存储 PCR 产品在4°c, 供以后使用。</li>
</ol>2. 聚丙烯酰胺凝胶铸件溶液的制备
<ol><li>在 dh2o 中溶解54克的三基和27.5 克硼酸, 加入20毫升 (pH 0.5), 用 dh8.0o 将溶液调整为1、000毫升的最终体积, 5×1升。 注: 缓冲器可在室温下储存数月。</li>
	<li>将200毫升的5×缓冲液添加到 dH2O 到最终容积2升, 并在室温下贮存, 0.5×2升的缓冲液。</li>
	<li>在0.5×缓冲液中溶解29克分子生物学级丙烯酰胺和1克分子生物学级 n、n '-methylenebisacrylamide, 制备6% 的非变性聚丙烯酰胺凝胶溶液, 最终体积为500毫升。用铝箔盖住溶液瓶, 贮存在4摄氏度, 供以后使用。 注意: 丙烯酰胺被认为是一种强有力的神经毒素, 应该小心处理。在称量粉末和处理包含它的解决方案时, 总是戴上手套。</li>
	<li>通过将2克 ap 与 dH2O 溶为10毫升的最终体积, 准备20% 过硫酸铵 (aps) 溶液。它可以储存在-20 °c 几个月。 注: 为方便起见, 10 毫升 20% APS 溶液可 aliquoted 为1.5 毫升或2毫升的离心管储存在-20 摄氏度。在使用前要解冻并混合好。</li>
	<li>将3µL 的束缚硅烷溶于0.997 毫升的95% 乙醇中, 制备稀释的束缚硅烷溶液, 其中含有0.5% 的乙酸。它可以储存在4摄氏度的几个星期。 注意: 粘合硅烷是有毒的, 在处理溶液时戴上手套, 保持房间通风。</li>
</ol>3. 电泳用聚丙烯酰胺凝胶的制备
<ol><li>在自来水中用洗涤剂清洗一组玻璃板和垫片, 然后用 dH2进行彻底冲洗, 然后将这些盘子放在板材烘干机架上晾干。</li>
	<li>将1毫升的束缚硅烷溶液分散到矩形玻璃板的内表面, 干燥5分钟。 注: 如果粘合-硅烷溶液不均匀地分布在板材表面, 凝胶在染色过程中可能被分离, 导致染色效果不理想。</li>
	<li>用拭子将1毫升的排斥硅烷分散到缺口玻璃板的内表面, 用纸巾擦拭多余的排斥硅烷, 空气干燥5分钟。 注: 如果排斥硅烷不均匀地涂层玻璃板材的表面, 它可能通过部分剥离损坏胶凝体。</li>
	<li>在顶部装配有缺口板的玻璃板, 并用铸件夹具将装配的两侧夹紧。</li>
	<li>倒入适当数量 (40 毫升) 6% 非变性聚丙烯酰胺凝胶溶液的烧杯为33厘米宽 x 10 厘米高度和间隔厚度1.5 毫米, 添加20µL 的 tetramethylethylenediamine (TEMED) 和200µLfresh 20% APS 和混合轻轻。 注: 凝胶溶液的适量 (40 毫升) 是从两个板的内部容积 (30 厘米 x 8.5 厘米 x 0.15 厘米) 计算出来的。对于凝胶聚合的 TEMED 和 aps 的用量, 根据参考17, 有一个标准的凝胶溶液与 TEMED 和 aps 的比例。上面描述的凝胶溶液储存在4°c。如果凝胶溶液贮存在室温下, 应使用20µL 的 TEMED 和100µL 20% APS。用玻璃棒轻轻搅动凝胶溶液的混合物, 以避免溶液中气泡。</li>
	<li>立即将溶液倒入切口板边缘的组装玻璃板上, 将空间几乎填满, 然后插入梳子。在梳子上加入一小块凝胶溶液。 注: 保持玻璃板水平, 防止凝胶泄漏。如有必要, 用气泡钩去除气泡。</li>
	<li>允许凝胶在室温下设置为30分钟。 注: 聚合时间可能随着凝胶溶液和环境温度的变化而改变。</li>
	<li>在凝胶完全聚合后, 取下铸件夹具, 将所设的板套放在电泳槽单元中, 并将缺口板朝向上缓冲储层, 用大钳将板材固定在油箱单元上。</li>
	<li>将 0.5×1升的缓冲液倒入上、下腔。用吸管或注射器取出梳子, 用缓冲器彻底冲洗所有水井。 注: 确保在盘子底部的凝胶三明治完全浸泡在没有气泡的缓冲液中。</li>
</ol>4. 运行凝胶
<ol><li>将1µL 的 PCR 产物载入聚丙烯酰胺凝胶的各个井中。将 DNA 大小的梯子和 PCR 产品的样品一起载入凝胶的两侧。将安全盖固定在上部缓冲室。</li>
	<li>将引线连接到电源, 将颜色编码的红色与红色和黑色相匹配。在110伏的恒定电压下运行凝胶, 直到染料达到定义的位置, 通常为 ~ 70 分钟。 注: 电压和运行时间可根据 PCR 产品的大小进行调整。</li>
</ol>5. 用于检测非聚丙烯酰胺凝胶中 SSR 标记的银染色法
<ol><li>电泳后, 将缓冲液从上部腔体排出到大烧杯中通过阀门。分离侧夹, 从设备上取下盘子, 小心地将缺口玻璃板沿一侧与刮刀分开, 并确保该凝胶仍然附着在涂有束缚硅烷的其它玻璃板上。</li>
	<li>1 l 的新鲜浸渍溶液溶解1.5 克 AgNO3在 dh 的 1 L 中2o. 通过溶出毫升的氢氧化钠, 在 dh 1 O, 添加 10 ml 900 毫升甲醛的情况下, 制备2升的新开发溶液., 并调整为使用 dH2O 的最终卷1升。 注: 佩戴手套并小心处理上述化学品及解决方案。没有必要在黑暗中保留解决方案和相应的步骤。</li>
	<li>用足够的 dH2O 仔细冲洗凝胶和玻璃板, 去除电泳缓冲液, 然后将该板与凝胶置于塑料托盘上, 将凝胶浸入浸渍液的1升中。把托盘放在振动筛上。</li>
	<li>轻轻摇动托盘在 60 r/分钟3-4 分钟。 注: 在浸渍溶液中, 可根据硝酸银的凝胶厚度和浓度来调节震动时间。</li>
	<li>把盘子移出浸渍液, 放到另一个托盘上。用足够的 dH2O 两次, 分别从板表面和凝胶中冲洗残余浸渍液, 每双3-5 秒。</li>
	<li>将印版与胶面置于另一托盘上, 将板材浸入1升的开发溶液中, 然后轻轻地摇动托盘 50 r/分钟3分钟. 监测 dna 片段的出现和停止发育当 dna 片段信号的最高比率 对背景噪声被观察 (这步采取 ~ 3 分钟)。</li>
	<li>用足够的 dH2O 两次冲洗盘子和凝胶, 用纸巾将凝胶板烘干。</li>
	<li>使用适当的扫描仪扫描凝胶。300 dpi 的分辨率给出了 DNA 片段的清晰可视化。这种凝胶也可以用照相机拍照。</li>
	<li>SSR 标记的带状模式可以根据图像中的 DNA 片段进行评分。</li>
</ol>

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作者声明他们没有竞争的财政利益。

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 ...

a-fast-silver-staining-protocol-enabling-simple-efficient-detection

Research

JoVE Journal

Genetics

10.7K 次观看.  Guangzhou University. 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。

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

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 ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...