期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2003
卷号：100
期号：26
页码：15440-15445
DOI：10.1073/pnas.2237126100
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 {varphi}X174 (5,386 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 {varphi}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; {approx}60 such segments would be required for a minimal cellular genome.
