A simple in vitro site directed mutagenesis of concatamerized cDNA by inverse polymerase chain reaction.
A simple in vitro site directed mutagenesis of concatamerized cDNA by inverse polymerase chain reaction.
复制标题
通过反向聚合酶链反应对多联 cDNA 进行简单的体外定点诱变。
DOI：
10.1093/nar/20.19.5241
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发表时间：
1992
影响因子：
14.9
通讯作者：
Galili,U
中科院分区：
文献类型：
--
作者：
Heda,GD;Henion,TR;Galili,U
定点诱变可以在质粒中通过反向聚合酶链式反应（PCR）用其中一个含有突变的“背靠背”引物进行。然后连接PCR产物以使质粒再环化用于进一步转化（1）。这种方法受到质粒大小的限制，因为在有核细胞中用作表达载体的质粒对于PCR来说通常太大。本文介绍了这种方法的一种改进，其中用多联体DNA进行反向PCR。因此，整个定点诱变程序在体外进行;不依赖于质粒，并且诱变的cDNA不含野生型分子。突变可以插入cDNA中的任何位点（图1）。这种简单的方法是潜在的有用的酶的催化结构域的识别，和各种生物活性蛋白质的功能结构域。全长cDNA分子用各自含有限制性位点的上游和下游引物A和Z合成（图1）。AZ PCR产物被限制酶切割并进行过夜连接。将连接产物在1%琼脂糖凝胶上电泳，并将多联体化的DNA分子收集到DEAE硝酸纤维素膜上。该DNA制备物不含单体cDNA，用作所有后续诱变反应的来源。将一纳克多联体化DNA用背靠背引物1和2进行反向PCR，引物1和2中的一个或两个含有所需突变。DNA相对链上的这些背对背引物可以对应于cDNA的任何区域。通过凝胶电泳分离1-2 PCR产物，以除去任何多联体化的野生型cDNA序列。该PCR产物通过Klenow DNA聚合酶进行末端冲洗，使得酶的核酸外切酶活性将去除任何非模板添加的碱基对（1）。随后，1-2 PCR产物在5 '端磷酸化，分子进行平端连接以形成多联体。使用引物A和Z对所得多联体进行第三次PCR。该AZ PCR产物具有与原始cDNA相同的序列，除了由引物1和/或2引入的突变。在将诱变的cDNA插入表达载体后，使用引物A和Z中的限制性位点，可以评估引入的突变对cDNA编码的蛋白质的影响。我们同时将两个突变引入糖基化酶α 1，3半乳糖基转移酶（α 1，3 GT）的cDNA（2）。从绒猴细胞系B 95制备cDNA。8.使用的引物根据它们在小鼠α 1，3GT cDNA中的位置编号（2），并具有以下序列：引物A：5’GCCGAATTCGAGATGGCAGAAGGACTGGT（碱基207-225）和引物Z：5’GCCGAATTCTTGAAGT-CAGACATTATTTCT（碱基1191-1171的互补序列）。两条引物在其5 ′端均含有EcoRI限制性位点。用VentT DNA聚合酶（试剂盒购自Promega Corp.，麦迪逊，威斯康星州）。用EcoRI切割AZ PCR产物。将5微克EcoRI切割的AZ PCR产物在总体积为20 μ L的含有3单位T4 DNA连接酶（GIBCO-BRL，盖瑟斯堡，MD）、15%（w/v）PEG-8000、25 mM Tris-HCl（pH 7.6）、5 mM MgCl 2、0.5 mM DTT和0.5 mM ATP的溶液中于18 ℃自连接16小时。将该连接产生的多联体电转移到NA 45 DEAE硝酸纤维素膜（Schleicher和Schuell）上（3）。这些...
Site directed mutagenesis can be performed in plasmids by inverse polymerase chain reaction (PCR) with'back to back'primers one of which contains the mutation. The PCR product is then ligated in order to recircularize the plasmid for further transformation (1). This method is limited by the size of the plasmid, since plasmids used as expression vectors in nucleated cells are often too large for PCR. A modification of this methods is presented here, where the inverse PCR is performed with concatamerized DNA. Thus the entire site directed mutagenesis procedure is performed in vitro; independent of plasmids, and the mutagenized cDNA is devoid of the wild type molecules. The mutations can be inserted at any site in the cDNA (Figure 1). This simple approach is potentially useful for the identification of catalytic domains in enzymes, and functional domains in various biologically reactive proteins. The full length cDNA molecule is synthesized with upstream and downstream primers A and Z each containing a restriction site (Figure 1). The AZ PCR product is cleaved by the restriction enzyme and subjected to overnight ligation. The ligated product is run on a 1% agarose gel and the concatamerized DNA molecules are collected onto a DEAE nitrocellulose membrane. This DNA preparation, which is devoid of the monomeric cDNA, serves as a source for all subsequent mutagenesis reactions. One nanogram of the concatamerized DNA is subjected to inverse PCR with the back to back primers 1 and 2, either one or both of which, containing the desired mutation (s). These back to back primers on opposite strands of DNA can correspond to any region of the cDNA. The 1-2 PCR product is isolated by gel electrophoresis in order to remove any concatamerized wildtype cDNA sequences. This PCR product is subjected to end flushing by Klenow DNA polymerase, so that the exonuclease activity of the enzyme will remove any nontemplate added basepairs (1). Subsequently, the 1-2 PCR product is phosphorylated at the 5'end and the molecules undergo blunt end ligation in order to form concatamers. The resulting concatamers are subjected to a third PCR using primers A and Z. This AZ PCR product has the same sequence as the original cDNA except for the mutation (s) introduced by primers 1 and/or 2. The effect of the introduced mutations on the protein encoded by the cDNA can be assessed after insertion of the mutagenized cDNA into an expression vector, using the restriction site in primers A and Z.We introduced simultaneously two mutations into the cDNA of the glycosylation enzyme al, 3galacytosyltransferase (al, 3GT)(2). The cDNA was prepared from the marmoset cell line B95. 8. The primers used were numbered according to their location in the mouse a I, 3GT cDNA (2), and had the following sequences: Primer A: 5'GCCGAATTCGAGATGGCAGAAGGACTGGT (bases 207-225) and Primer Z: 5'GCCGAATTCTTGAAGT-CAGACATTATTTCT (complementary sequence of bases 1191-1171). Both primers contained an EcoRI restriction site at their 5'end. This and subsequent PCR reactions were performed with VentT DNA polymerase (kit purchased from Promega Corp., Madison, WI). The AZ PCR product was cleaved with EcoRI. Five micrograms of the EcoRI cleaved AZ PCR product were self ligated in a total volume of 20 IL containing 3 units of T4 DNA ligase (GIBCO-BRL, Gaithersburg, MD), 15%(w/v) PEG-8000, 25 mM Tris-HCI (pH 7.6), 5 mM MgCl2, 0.5 mM DTT and 0.5 mM ATP at 18-C for 16 hours. The concatamers resulting from this ligation were electrotransferred on NA45 DEAE nitrocellulose membrane (Schleicher and Schuell)(3). These …