Smith Hamilton O, Hutchison Clyde A, Pfannkoch Cynthia, Venter J Craig
Institute for Biological Energy Alternatives, 1901 Research Boulevard, Suite 600, Rockville, MD 20850, USA.
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15440-5. doi: 10.1073/pnas.2237126100. Epub 2003 Dec 2.
We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (i). gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii). ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii). assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule. We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification. PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements. The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp. However, fully infectious X174 virions were recovered after electroporation into Escherichia coli. Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes. One such isolate had exactly the intended sequence. We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; approximately 60 such segments would be required for a minimal cellular genome.
我们改进了方法，大幅缩短了从合成寡核苷酸精确组装5至6 kb DNA片段所需的时间。作为对该方法的测试，我们已确定了从一组化学合成寡核苷酸快速（14天）组装噬菌体X174完整感染性基因组（5386 bp）的条件。该过程包括三个关键步骤：（i）对混合寡核苷酸进行凝胶纯化，以减少不正确链长分子的污染；（ii）在严格退火条件（55℃）下连接寡核苷酸，以防止不正确序列分子的退火；（iii）通过聚合酶循环组装将连接产物组装成全长基因组，这是一种非指数反应，其中每个末端寡核苷酸只能延伸一次以产生全长分子。在对聚合酶循环组装产物进行凝胶分析时，我们观察到了全长组装的离散条带，无需任何PCR扩增。然后使用PCR扩增获得大量纯全长基因组用于环化和感染性测量。合成DNA的感染性低于天然DNA，表明每500 bp约有一个致死错误。然而，将其电穿孔导入大肠杆菌后可回收完全具有感染性的X174病毒粒子。对几个感染性分离株的序列分析验证了这些合成基因组的准确性。其中一个分离株具有完全预期的序列。我们建议通过连接单独组装的5至6 kb片段来组装更大的基因组；最小细胞基因组大约需要60个这样的片段。
