AN OLIGODEOXYRIBONUCLEOTIDE DIRECTED DUAL AMBER METHOD FOR SITE-DIRECTED MUTAGENESIS
AN OLIGODEOXYRIBONUCLEOTIDE DIRECTED DUAL AMBER METHOD FOR SITE-DIRECTED MUTAGENESIS
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DOI：
10.1016/0378-1119(94)00750-m
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发表时间：
1995-01-23
期刊：
影响因子：
3.5
通讯作者：
NAKAGAWA, M
中科院分区：
文献类型：
--
作者：
HASHIMOTOGOTOH, T;MIZUNO, T;NAKAGAWA, M
利用两个寡脱氧核苷酸引物建立了一种简单的体外定点突变方法。一个用作选择引物，另一个用于靶DNA中的诱变（诱变引物）。突变克隆可以通过cat基因中双琥珀（am）密码子到双Gln密码子的回复突变来选择，从而在sup（0）宿主细胞中产生氯霉素抗性表型。与以前报道的方法不同，新方法既不需要特殊的化学试剂，也不需要单链DNA纯化，也不需要任何额外的生化处理，如多重PCR、限制性消化等，而是利用寡核苷酸定向的双链扩增（ODA）质粒之一，即，pKF 16 c、pKF 17 c、pKF 18 c或pKF 19 c。在cat（cat（am 2））中位于彼此相隔两个密码子的两个琥珀（am）突变可以在引物延伸期间用20个核苷酸的引物（CQ，pAACCAGACCGTTCAGCTGGA）同时回复。在使用lacZ'基因的模型实验中，使用编码lacZ'的am或sbd的诱变引物和cat（am 2）的CQ引物的混合物，经常在lacZ'中共引入am突变或单bp缺失（sbd）突变。将该方法应用于人AT cDNA（编码抗凝血酶），将涉及先天性血栓形成疾病的人AT变体之一AT-Kyoto中的错义突变（Arg（406）-> Met）有效地引入野生型cDNA（>85%）。
A simple procedure for in vitro site-directed mutagenesis (SDM) was developed using two oligodeoxyribonucleotide (oligo) primers. one serving as selection primer and the other for mutagenesis in the target DNA (mutagenic primer). The mutant clones can be selected by reversion mutations of dual amber (am) to dual Gln codons in the cat gene, resulting in a chloramphenicol-resistant phenotype in sup(0) host cells. Unlike previously reported procedures, the new method requires neither special chemical reagents, nor single-stranded DNA purification, nor any additional biochemical treatments such as multiple PCRs, restriction digestion and so on, but utilises one of the oligo-directed dual am (ODA) plasmids, i.e., pKF16c, pKF17c, pKF18c or pKF19c. The two amber (am) mutations located two codons apart from each other in cat (cat(am2)) can be simultaneously reverted with a 20-nucleotide primer (CQ, pAACCAGACCGTTCAGCTGGA) during primer extension. In a model experiment using the lacZ' gene, an am mutation or a single-bp deletion (sbd) mutation was frequently co-introduced in lacZ' using a mixture of am- or sbd-encoding mutagenic primer for lacZ', and the CQ primer for cat(am2). Applying this procedure to the human AT cDNA (encoding antithrombin), a missense mutation (Arg(406) --> Met) in one of the human AT variants, AT-Kyoto, involved in congenital thrombosis disease, was introduced efficiently into the wild-type cDNA (>85%).