SINGLE-STEP ASSEMBLY OF A GENE AND ENTIRE PLASMID FROM LARGE NUMBERS OF OLIGODEOXYRIBONUCLEOTIDES
SINGLE-STEP ASSEMBLY OF A GENE AND ENTIRE PLASMID FROM LARGE NUMBERS OF OLIGODEOXYRIBONUCLEOTIDES
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DOI：
10.1016/0378-1119(95)00511-4
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发表时间：
1995-10-16
期刊：
影响因子：
3.5
通讯作者：
HEYNEKER, HL
中科院分区：
文献类型：
--
作者：
STEMMER, WPC;CRAMERI, A;HEYNEKER, HL
在这里，我们描述了装配聚合酶链式反应是一种从大量的寡聚脱氧核糖核苷酸(寡核苷酸)合成长DNA序列的方法。这种方法源于DNA改组[Stemmer，Natural 370(1994a)389-391]，它不依赖于DNA连接酶，而是依赖DNA聚合酶在组装过程中构建越来越长的DNA片段。在一次反应中，从56个寡核苷酸中组装了一个1.1kb的含有TEM1β-内酰胺酶编码基因(BlA)的片段，每个寡核苷酸长40个核苷酸(NT)。以四环素抗性基因(TC-R)为唯一可选择标记，对合成基因进行了PCR扩增，并克隆到载体中。在不依赖氨苄西林(AP)选择的情况下，76%的TC-R克隆是AP(R)，这使得这种方法成为快速和经济有效地合成任何基因的通用方法。我们通过在一个包含134个寡核苷酸的单一反应容器中合成一个包含bla基因、lacZ基因的α片段和复制的PUC起始点的2.7kb高分子质量多聚体来测试组装PCR的范围。用一种独特的限制性内切酶消化，然后连接并转化到大肠杆菌中，产生了正确的质粒。装配聚合酶链式反应非常适合于几种体外诱变策略。
Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplifed and cloned in a vector containing the tetracycline-resistance gene (Tc-R) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the Tc-R colonies were Ap(R), making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.