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克隆基因全长读码框的秘诀

发布: 2018-10-09 8,427阅读 687评论

在分子克隆实验中，获取基因的全长开放读码框（ORF）有时候会是一个挑战。对于新手来说，这个实验有时候可以卡很久。但这绝对不是传说中看脸的实验，而是有很多方法将成功率堆到近乎100%的。这篇文章将为大家讲解获取全长ORF的一些秘诀。

不，你听我解释……真不是上面那样子的……

选择合适的起始材料
首先应该明确，我们的实验目的是获取ORF，而不要引入其他的复杂因素。因此，我们应该选一些好抽提RNA的材料来做实验（比如培养的细胞），而不是非得跟自己过不去，偏偏选择一些难处理的样本（比如组织样品，还是难搞的那种）。

其次，要查清楚所选择的材料中，目的基因的表达量有多高。比如说，我们要克隆GFAP基因。这个基因主要在脑星形胶质细胞中表达，而在其他类型的细胞中表达量极低，甚至不表达。因此，如果要克隆这个基因，就务必使用胶质细胞来提取RNA。此外，GFAP基因在一些病理情况下表达会降低，比如在一些胶质瘤细胞中就不表达了。所以，在正式动手之前，一定要好好调查手头的原材料到底有没有表达目的基因。

基因表达量的信息去哪里查呢？如果是人类样本的话，这里推荐两个数据库：The Human Protein Atlas（HPA，http://www.proteinatlas.org/）和GTEx（https://www.gtexportal.org/home/）。只要输入基因的名字，就可以看到这个基因在各组织器官的表达水平了。其中，HPA整合了一部分GTEx的数据。所以一般来讲，查一个就够了。

而如果是研究其他物种，暂时来说，最好是去查专门的物种基因表达数据库。另外也可以试试这个数据库：Expression Atlas（https://www.ebi.ac.uk/gxa/home/），可以检索基因不同物种在不同部位的表达情况。

抽提高质量的RNA
要扩增出全长的ORF，首先要保证一开始提取的RNA的完整性。涉及RNA的实验，往往要注意所使用的器具、试剂、耗材不含有RNA酶（RNase）。同时，也得注意周遭的环境，找一个干净整洁的地方来操作。

不过，要防止RNase的污染，并没有想象中的那么复杂，也不是像传说中那样，器具耗材需要泡DEPC、实验者要戴口罩等。之前的一篇文章，专门探讨这个问题：实验 | RNA提取实验中的那些玄学

简单来说，常规的高温高压灭菌已可认为灭活了RNase。而吸头与离心管可以直接购买无核酸酶认证的产品。在实验过程中，也无需特别注意低温操作。我现在用QIAGEN的RNA抽提试剂盒，全程都是室温操作，包括裂解、离心、洗涤等步骤，从未发生过RNA降解的情况。

逆转录引物
逆转录实验所使用的引物包括了Oligo dT，随机六聚体（Random hexamer）和基因特异引物（GSP）三种。每一种引物，都有其特定的适用范围。比如说，如果合成的cDNA是要用来做基因表达量研究的，一般建议使用Anchored Oligo dT（3’端带有两个锚定碱基），保证逆转录产物的能够代表原始RNA的丰度。但如果是要拿全长ORF，在引物的选择上就有所讲究。

常用的Oligo dT引物可以选择性地将含有poly A尾巴的RNA——即所有的蛋白质编码RNA和部分长链非编码RNA，给逆转录出来。但是，逆转录酶沿着逆转录引物从5’->3’端一路合成过去，是需要“翻山越岭”的。因为，RNA的三级结构非常复杂，并不是老老实实的一条线性的单链，而是会形成各种茎环结构和其他折叠结构。因此，逆转录酶往往会跑到一半就卡住跑不动，然后从模板链上掉下来，cDNA合成到一半也就中止了。这就导致了，Oligo dT所拿到的cDNA具有3’端偏好性。（见下图）

Hexamer对整个转录组的覆盖度是最高的。因为它不需要RNA模板携带特定序列，也不需要像Oligo dT引物一般从RNA的末端开始逆转录，而是可以好几条Hexamer贴到一条RNA链的各个部位，直接引导逆转录的进行。不过，由于逆转录依然是从引物的5’->3’端进行。如果某个RNA模板的末端（即3’端）没有Hexamer的结合，它的末端就无法被逆转录成cDNA。也就是说，Hexamer所拿到的cDNA具有5’端偏好性。（见下图）

怎么办？貌似挑哪种引物都不好啊。解决方法超级简单，只需在逆转录时，把Oligo dT和Hexamer都加进去反应体系就好了。

使用耐高温H minus逆转录酶
目前市售的逆转录酶，有AMV和MMLV两种来源。以前，实验者还需要为选择哪一种逆转录酶头痛。因为在比较“古早”的时候，这两种酶有各自明显的优势和缺陷：AMV逆转录酶耐高温，而高温有助于打开RNA的高级结构从而有利于反应进行，然而携带RNase H活性，会边逆转录边降解RNA模板，导致产率低，cDNA长度普遍在5 kb左右。而MMLV逆转录酶的RNase H活性低，但不耐高温，无法逆转录具有高级结构的RNA。

现在的实验者则幸福多了，完全不需要烦恼这些问题。得益于对MMLV逆转录酶的工程改造，我们已经获得了一些移除RNase H活性，提高热稳定性但又保留逆转录活性的突变体，并制成了商品化试剂。其中有性能优异的Invitrogen SuperScript系列，但价格也很感人，不到特殊的珍贵样品我不拿出来用。一般情况下，常规的耐高温H minus逆转录酶（比如我自己选用的Thermo Scientific Maxima H Minus），就能满足需求。这些酶能在50℃甚至更高的温度下催化反应进行，而这样的高温已经足以打开绝大多数RNA的高级结构了。

性能很强悍没有之一的SuperScript IV，但价格非常感人……

所以我一般用这个，甚至不买kit只买酶，剩下dNTP和RT引物都可以去合成，便宜到掉渣。当然你可选用任何惯用的产品。

用组装的方法获得长链DNA
哺乳动物平均mRNA长度略大于2,000 nt，微生物的会更短一些。但是，有一些奇葩的基因，其mRNA的长度会突破天际。比如Titin（TTN）基因的转录本是人类最长的mRNA，全长108,861 nt。我以前曾经碰过一个CBP（CREBBP）基因，转录本破10 k碱基。

用PCR的方法，从cDNA那里一次性扩出10 kb甚至更长的片段，往往是非常困难的。这不光是PCR效率的问题。在一管逆转录产物中，完整性那么好的cDNA模板，数量也不会很多。就算是欧皇，电泳看到条带了，但一测序可能会发现好几个突变，这种情况真的会让人很想死。

不过，现在DNA片段拼接试剂盒已经非常成熟。假如一次性拉不出那么长的片段，那就用几条短片段给组装出来。如果大家有兴趣，我可以再写一篇文章，介绍Gibson assembly技术。

目前，全基因合成服务公司所采用的技术路线，也是先合成小片段再拼接的。

Gibson assembly是一种能将多个小片段无缝、定向组装成大片段的技术

总结
话说……如果Addgene有得卖的话，那直接去买算了……

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