Agarose Gel Electrophoresis for the Separation of DNA Fragments
Agarose Gel Electrophoresis for the Separation of DNA Fragments
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DOI：
10.3791/3923
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发表时间：
2012-04-01
影响因子：
1.2
通讯作者：
Kim, Yong Hoon
中科院分区：
文献类型：
--
作者：
Lee, Pei Yun;Costumbrado, John;Kim, Yong Hoon
琼脂糖凝胶电泳是分离大小从100 bp到25 kb不等的DNA片段的最有效方法（1）。琼脂糖分离自石花菜属和江蓠属海藻，由重复的琼脂二糖（L-和D-半乳糖）亚基组成（2）。在凝胶化过程中，琼脂糖聚合物以非共价键结合并形成束的网络，其孔径决定凝胶的分子筛性质。琼脂糖凝胶电泳的使用彻底改变了DNA的分离。在采用琼脂糖凝胶之前，DNA主要使用蔗糖密度梯度离心分离，其仅提供近似大小。为了使用琼脂糖凝胶电泳分离DNA，将DNA加载到凝胶中的预制威尔斯孔中并施加电流。DNA（和RNA）分子的磷酸骨架带负电荷，因此当置于电场中时，DNA片段将迁移到带正电荷的阳极。由于DNA具有均匀的质荷比，DNA分子在琼脂糖凝胶中以一定的模式按大小分离，使得行进的距离与其分子量的对数成反比（3）。DNA在琼脂糖凝胶中移动的主要模型是“偏向爬行”，即前沿向前移动并拉动分子的其余部分沿着（4）。DNA分子通过凝胶的迁移速率由以下确定：1）DNA分子的大小; 2）琼脂糖浓度; 3）DNA构象（5）; 4）施加的电压，5）溴化乙锭的存在，6）琼脂糖的类型和7）电泳缓冲液。分离后，DNA分子可以在用适当的染料染色后在紫外光下可视化。通过遵循本协议，学生应该能够：1。了解DNA片段在凝胶基质中分离的机制2。了解DNA分子的构象如何决定其在凝胶基质中的移动性3。确定适合他们需要的琼脂糖溶液浓度4.准备琼脂糖凝胶用于DNA样品的电泳5。设置凝胶电泳仪和电源6.选择合适的电压用于分离DNA片段7。了解溴化乙锭
Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to:1. Understand the mechanism by which DNA fragments are separated within a gel matrix2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix3. Identify an agarose solution of appropriate concentration for their needs4. Prepare an agarose gel for electrophoresis of DNA samples5. Set up the gel electrophoresis apparatus and power supply6. Select an appropriate voltage for the separation of DNA fragments7. Understand the mechanism by which ethidium bromide