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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