在之前的文章中，小编给大家简介了四种提纯DNA的方法（历史文章点这里），分别是苯酚/氯仿抽提法、乙醇沉降法、硅胶柱吸附法和磁珠吸附法。其中，关于硅胶柱吸附法，也有过详细介绍（点这里），那么，今天小编就来说说苯酚抽提法。
苯酚抽提法非常简单，而且十分常见。特别是在提取基因组DNA时，要去掉许多从细胞裂解液中带来的蛋白，苯酚法十分好用。
苯酚抽提的基本步骤
- 将同体积的苯酚和（DNA+蛋白）混合物溶液混合；
- 苯酚和水溶液不互溶，所以分层，水层在上，苯酚层在下。快速震荡，让两层充分混合；
- 静置让两液层分开，分离水层，在水层中得到的应该是大量的DNA和可能少量的蛋白质。
基本原理
最核心的原理是水和苯酚作为有极性差异的溶剂，对DNA和蛋白质有不同的溶解能力。因为水分子中的氧原子有更强的电负性，共用电子对向氧原子偏离，产生了看似略带负电的氧原子端和略带正电的氢原子端，所以我们说水是很有极性的溶剂（如下图）。苯酚也有极性，但不像水分子那么明显，因为苯环也有电负性，所以苯酚的氧原子并不能太强吸引周围的电子（如下图）。
有一点我们是清楚的，DNA可溶于水，原因就是DNA也是有极性的分子（如下图，DNA分子的糖-磷骨架上的磷酸基团是带负电的）。对于一物质是否水溶，我们基本可以总结出：有极性（polar）=亲水性（hydrophilic）=可溶；无极性（non-polar）=憎水性（hydrophobic）=不溶
还有一点我们也是清楚的，细胞液环境是水环境，里面到溶解着各种蛋白质。蛋白质作为长链氨基酸，在形成有效结构的时候，外表面多是有极性的亲水类氨基酸（如谷氨酸、Glu；赖氨酸、Lys和组氨酸、His），内核多是没那么有极性的憎水类氨基酸，所以能够稳定的存在于水溶液中。
但是，在苯酚抽提法中，蛋白质在水中的溶解度被改变了。水溶液作为极性溶剂，导致了蛋白质的极性氨基酸在外，而没那没有极性的氨基酸在内；苯酚的到来，使得这些没那么有极性的氨基酸有了出头之日，他们被翻了出来，而那些有极性的氨基酸则被塞进蛋白质内核，以适应苯酚溶剂（基本原理如下图所示）。这样一来，变性的蛋白进入苯酚层，DNA则停留在水层。这也是为什么，有机溶剂会让蛋白质变性，因为不可逆地破坏了蛋白的整体结构。
苯酚抽提的过程和蛋白质变性原理
苯酚抽提，简单有效，只是苯酚有毒，操作的时候要小心哦！小伙伴们对DNA提纯有想法或心得，请在下方留言，欢迎讨论！
图片来自网络，其余文字部分为BioEngX原创，转载请注明！
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