在分子生物实验室中,我们常用各种试剂盒(e.g. QIAGEN mini/midi/maxi prep)做质粒DNA提取(质粒提取相关文章),如果都遵循操作,所得样本基本上是质粒DNA(plasmid),只有少量的基因组DNA(genomic DNA)会混入进来。那么问题来了,基因组DNA和质粒DNA同为DNA分子,为什么基因组DNA没有被纯化出来呢?难道硅胶柱都自己有意识不成,可以分辨哪个是小型环状DNA,哪个是大号基因组DNA……说到这里不得不提提取质粒DNA和基因组DNA的区别。
基因组DNA提取
相比于质粒DNA提取,基因组DNA的提取更简单些,步骤如下:
1裂解
只要打开细胞将基因组DNA释放出来就好了。一般来说,如果有细胞壁,就先破坏细胞壁,然后破坏细胞膜,细胞的DNA物质就全释放出来。关于打破细胞壁的方法,请参见历史文章(细胞裂解简述和五种细胞壁结构)。从经验上来看,机械裂解(如滚珠搅拌法和超声波破碎法)要比化学裂解(如酶溶裂解)要来得快而且全面。
2纯化
只要基因组DNA从细胞里释放出来,接下来要做的就是纯化它们。一般可以采用苯酚-氯仿抽提法(历史文章点这里)或者直接过硅胶柱。值得注意的是,基因组DNA由于很大,基本上在提纯以后不会保持完整,而是会碎裂成片段。但这对后续可能进行的PCR、qPCR不但没有坏处,反而是有好处的。不完整的DNA更容易变性,因此有益于PCR的进行。
质粒DNA提取
质粒DNA的提取比基因组DNA提取更复杂一些,因为要在提取的过程中避免基因组DNA的污染。和基因组DNA提取法最大的区别在于细胞裂解的方法。
1裂解
提取质粒DNA时,只采用强碱裂解法(alkaline lysis)。这个方法是由Birnboim和Doly在1979年发明的,在SCI上原著文章的引用量至今为止已达到12886,可以算是分子生物学里殿堂级的实验方法(科普ps:另外一个殿堂级方法,bradford assay,原文发于1976,至今引用量187783)。强碱裂解液里有氢氧化钠(NaOH)和十二烷基硫酸钠(SDS:Sodium dodecyl sulfate),目的是让质粒DNA和基因组DNA完全失活,使双链分子全部变成单链。使用强碱裂解的时候要注意不能裂解时间过长,否则对质粒DNA和基因组DNA产生不可逆形变就不好了(< 5 min)。
接下来强碱液被中和使裂解反应停止,这里就是分离质粒DNA和基因组DNA的关键。质粒DNA分子相对较小,互补链很容易重新组合成双链而回复活性,而基因组DNA较大,在形成双链的过程中很容易和其他蛋白缠绕在一起,导致无法形成完整的双链。和蛋白缠绕的基因组DNA就可以在提纯过程中被除去。
2提纯
可以用乙醇提取法或是硅胶柱提取(历史文章点这里)。这样提取出来的DNA基本上是质粒,而且可以用于克隆、测序等等后续的活动,如果质粒DNA要用于转染(transfection)或者离子交换(anion exchange),质粒还需要额外处理。
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