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[什么是桑格测序？](https://www.sigmaaldrich.com#what) - [桑格测序如何工作？](https://www.sigmaaldrich.com#how) - [桑格测序步骤](https://www.sigmaaldrich.com#sanger) - [How to Read Sanger Sequencing Results](https://www.sigmaaldrich.com#read) - [Sanger Sequencing vs.PCR](https://www.sigmaaldrich.com#sequencing) - [相关产品](https://www.sigmaaldrich.com#%E4%BA%A7%E5%93%81) ## [](https://www.sigmaaldrich.com)什么是桑格测序？ 桑格测序又称 "链终止法"，是一种确定 DNA 核苷酸序列的方法。该方法由两届诺贝尔奖获得者弗雷德里克-桑格（Frederick Sanger）和他的同事于 1977 年开发，因此被称为 桑格测序法。 回顾 DNA 的一般结构，请参阅 __图 2__。 ## [](https://www.sigmaaldrich.com)桑格测序是如何进行的？ 桑格测序可以手动进行，或者更常见的是通过测序机自动进行（__图 1__）。每种方法都遵循以下三个基本步骤。 ![自动桑格测序](https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/images/technical-documents/protocols/genomics/sequencing/sanger-sequencing_steps_process_diagram/sanger-sequencing_steps_process_diagram.png "自动桑格测序") __图 1.__桑格测序自动化的三个基本步骤。 ## [](https://www.sigmaaldrich.com)桑格测序步骤 桑格测序有三个主要步骤。用于链终止 PCR 的 DNA 序列 感兴趣的 DNA 序列被用作一种特殊类型的 [PCR](https://www.sigmaaldrich.com/SG/zh/technical-documents/technical-article/genomics/pcr/routine-amplification) 称为链终止 PCR。链终止 PCR 的工作原理与标准 PCR 相似，但有一个主要区别：添加了称为双脱氧核苷酸 (ddNTP) 的修饰核苷酸 (dNTP)。在标准PCR的延伸步骤中，DNA聚合酶通过催化最后一个核苷酸的游离3'-OH基团与下一个核苷酸的5'-磷酸之间形成磷酸二酯键（__图2__），将dNTPs添加到生长的DNA链中。 在链终止 PCR 中，用户在 PCR 反应中将低比例的链终止 ddNTP 与普通 dNTP 混合。ddNTP 缺少磷酸二酯键形成所需的 3'-OH  基团；因此，当 DNA 聚合酶随意加入一个 ddNTP 时，延伸就会停止。链终止 PCR 的结果是数百万到数十亿个相关 DNA 序列的寡核苷酸拷贝，以 5'-ddNTPs 的随机长度（n）终止。 在手动桑格测序中，需要设置四个 PCR 反应，每个反应只混合一种 ddNTP（ddATP、ddTTP、ddGTP 和 ddCTP）。 在桑格测序自动化过程中，所有 ddNTP 都混合在一个反应中，四种 dNTP 中的每一种都有一个独特的荧光标签。 ### 2.通过凝胶电泳进行大小分离 第二步，通过凝胶电泳按大小分离链端寡核苷酸。在凝胶电泳中，DNA 样品被装入凝胶基质的一端，然后施加电流；DNA 带负电，因此寡核苷酸会被拉向凝胶另一侧的正电极。由于所有 DNA 片段的单位质量电荷相同，因此寡核苷酸的移动速度只取决于其大小。片段越小，在凝胶中移动时受到的摩擦就越小，移动速度就越快。因此，寡核苷酸将从小到大排列，从下到上读取凝胶。 在手工桑格测序中，来自四个 PCR 反应的寡核苷酸分别在凝胶的四个泳道中运行。 在 自动 桑格测序中，所有寡核苷酸都在测序机内的单个毛细管凝胶电泳中运行。 ### 3.凝胶分析& DNA序列的确定 最后一步是读取凝胶，确定输入 DNA 的序列。由于 DNA 聚合酶只能从提供的引物开始按 5' 到 3' 的方向合成 DNA，因此每个末端 ddNTP 都对应于原始序列中的一个特定核苷酸（例如，最短的片段必须终止于 5' 到 3' 端）、最短的片段必须终止于 5' 端第一个核苷酸，第二短的片段必须终止于 5' 端第二个核苷酸，等等）。因此，通过读取凝胶条带从小到大的顺序，我们可以确定原始 DNA 链的 5' 至 3' 序列。 在 手动 桑格测序中，用户从下往上同时读取凝胶的所有四条泳道，利用泳道来确定每条条带末端 ddNTP 的身份。例如，如果在与 ddGTP 相对应的一列中发现了底部条带，那么最小的 PCR 片段以 ddGTP 终止，原始序列 5' 端第一个核苷酸是鸟嘌呤 (G) 碱基。 在桑格自动测序中，计算机依次读取毛细管凝胶中的每个条带，利用荧光来确定每个末端 ddNTP 的身份。简而言之，激光激发每个条带中的荧光标签，计算机检测由此发出的光。由于四种 ddNTP 都带有不同的荧光标签，因此发出的光可以直接与末端 ddNTP 的身份联系起来。输出结果称为色谱图，它显示了模板 DNA 长度方向上每个核苷酸的荧光峰。 ![DNA 结构示意图。DNA 是由两条相互缠绕形成双螺旋的链组成的分子。每条链都由一串称为脱氧核苷酸（dNTPs）的分子组成。](https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/images/technical-documents/protocols/genomics/sequencing/sanger-sequencing_dna-structure/sanger-sequencing_dna-structure.png "DNA 结构示意图。DNA 是由两条相互缠绕形成双螺旋的链组成的分子。每条链都由一串称为脱氧核苷酸（dNTPs）的分子组成。") __图 2.__DNA 结构示意图。DNA 是由两条相互缠绕形成双螺旋的链组成的分子。每条链都由一串称为脱氧核苷酸（dNTPs）的分子组成。 每个 dNTP 都含有一个磷酸基团、一个糖基和四个含氮碱基\[腺嘌呤 (A)、胸腺嘧啶 (T)、鸟嘌呤 (G) 或胞嘧啶 (C)]中的一个。通过一个 dNTP 的糖基和下一个 dNTP 的磷酸基之间的磷酸二酯共价键，dNTP 以线性方式串联在一起；这种重复的糖-磷酸模式构成了糖-磷酸骨架。 两条独立链上的含氮碱基通过互补碱基之间的氢键结合在一起，形成双链 DNA 螺旋。 ## [](https://www.sigmaaldrich.com)如何读取桑格测序结果 如何正确读取桑格测序结果取决于两条互补 DNA 链中哪一条是我们感兴趣的，以及有哪些引物。如果 DNA 的两条链分别是 A 和 B，且 A 链是我们感兴趣的，但引物更适合 B 链，那么输出片段将与 A 链相同；另一方面，如果 A 链是我们感兴趣的，引物更适合 A 链，那么输出结果将与 B 链相同。 因此，如果感兴趣的序列为 "TACG"，而引物最适合该链，那么输出将为 "ATGC"，因此必须转换回 "TACG"。但是，如果引物更适合互补链（"ATGC"），那么输出将是 "TACG"，这才是正确的序列。 简而言之，在开始之前，您需要知道您的目标是什么，以及如何达到目标！因此，请牢记这一点，下面是前一个示例（TACG -> ATGC -> TACG）。如果二脱氧核苷酸的标签是 T = 黄色，A = 粉红色，C = 深蓝色，G = 浅蓝色，那么最终会得到引物-A、引物-AT、引物-ATG 和引物-ATGC 这几个短序列。通过电泳分离片段后，激光会按照长度顺序（粉色、黄色、浅蓝色和深蓝色）读取片段，并生成色谱图。计算机将对字母进行转换，因此最终的序列就是正确的 TACG。 ## [](https://www.sigmaaldrich.com)桑格测序与 PCR 的比较 桑格测序与 PCR 的比较？ Sanger 测序和 PCR 使用类似的起始材料，可以结合使用，但两者都不能取代对方。 PCR 用于扩增整个 DNA。虽然可能会意外产生不同长度的片段（例如，DNA 聚合酶可能会脱落），但我们的目标是复制整个 DNA 序列。为此，"成分 "包括目标 DNA、核苷酸、DNA 引物和 DNA 聚合酶（特别是 Taq 聚合酶，它能在 PCR 所需的高温下存活）。 相比之下，桑格测序的目标是生成目标 DNA 的所有可能长度，直至目标 DNA 的全长。这就是为什么除了 PCR 起始材料外，还需要双脱氧核苷酸的原因。 在为桑格测序方案生成起始材料时，桑格测序和 PCR 可以结合使用。 如果有一个以上的模板，桑格测序就会更有效。如果目标序列长达 1,000 个核苷酸，而模板只有一个拷贝，那么生成 1,000 个标记片段将需要更长的时间。但是，如果模板有多个拷贝，理论上生成全部 1000 个标记片段所需的时间会更短。 ## 相关产品 抱歉，发生了意外错误 Response not successful: Received status code 500 __相关文章__ - 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