Total synthesis of multi-kilobase DNA sequences from oligonucleotides
Total synthesis of multi-kilobase DNA sequences from oligonucleotides
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DOI：
10.1038/nprot.2006.426
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Santi, Daniel V.
中科院分区：
文献类型：
--
作者：
Reisinger, Sarah J.;Patel, Kedar G.;Santi, Daniel V.
说明了从40-mer寡核苷酸合成DNA的方法，我们使用该方法生成32-kb的DNA片段。在两步PCR反应中将DNA序列合成为类似于500 bp片段（双链体），并使用非连接依赖性克隆（LIC）进行克隆。然后以逐步的方式将合成子组装成更长的全长序列。通过最初合成较小的片段（多核苷酸），与在单个步骤中合成完整的多核苷酸酶DNA序列相比，测序的克隆的数量较低。LIC消除了克隆前纯化片段的需要，使该过程适合高通量操作和自动化。IIs型限制性内切酶允许双链体的无缝组装，而不限制合成的序列。使用允许选择具有两个独特抗生素抗性标记的所需克隆的载体，成对组装合成片段以产生最终构建体，并且这消除了在用限制性内切核酸酶消化后纯化片段的需要。
A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as similar to 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.