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Generating a Synthetic Genome by Whole Genome Assembly: ϕX174 Bacteriophage from Synthetic Oligonucleotides
Hamilton O. Smith, Clyde A. Hutchison III, Cynthia Pfannkoch and J. Craig Venter
Proceedings of the National Academy of Sciences of the United States of America
Vol. 100, No. 26 (Dec. 23, 2003), pp. 15440-15445
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3149024
Page Count: 6
You can always find the topics here!Topics: Oligonucleotides, Genomes, Ligation, Molecules, DNA, Polymerase chain reaction, Principal components analysis, Genetic mutation, Bacteriophages, Sequencing
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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 ϕX174 (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°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 ϕX174 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; ≈60 such segments would be required for a minimal cellular genome.
Proceedings of the National Academy of Sciences of the United States of America © 2003 National Academy of Sciences