Humpty Dumpty had a famous problem — he sat on a wall, had a great fall and...couldn't be put back together again. Genome biologists have had a similar problem until now — they could break genomes up and sequence them but not synthesize them de novo, or at least not in any efficient way. A group led by Craig Venter has now devised a method for assembling the complete genome of a bacteriophage from a pool of chemically synthesized oligonucleotides. Although still in need of a correcting mechanism, the method might in future be used to make synthetic chromosomes.

Driven by the desire to improve on previous attempts to assemble active viral genomes, Smith et al. chose the φX174 bacteriophage — the first living organism to have its 5,386 bp genome sequenced — to test their three-step approach to synthetic genome assembly. The first step involves oligonucleotide synthesis, based on both strands of the phage genome. In the second step, the oligonucleotides are phosphorylated, mixed and ligated to give, on average, 700-bp double-stranded fragments. Two rounds of polymerase cycle assembly are used in the third step to assemble these fragments into full-length genome molecules, which are PCR-amplified, linearized and tested for size by electrophoresis.

As the ultimate test of successful in vitro synthesis is that of molecular function, recircularized synthetic phages were electroporated into Escherichia coli to test for their infectivity. Careful comparison of infection efficiency between the wild-type and the synthetic phages indicated that some 9–10 inactivating mutations were present per synthetic genome, although at least one synthetic isolate was 100% identical to the original φX174 sequence. Smith et al. suggest that these point mutations probably result either from replication errors or are already present in the oligonucleotides.

Given the mutation rate, the authors point out that without an ability to select for the wild-type sequence, synthetic forms will carry, on average, a point mutation per 2 kb. Undeterred by this, they propose that, in combination with DNA sequencing and site-directed repair, their method can be used to assemble larger genomes.

Synthetic genomics could solve the problems of dealing with degraded biological material or extinct species. Genome sequences could also be modified during synthesis to provide an alternative to genetic engineering. Although this application of synthetic genomics might be around the corner for microbial genomes, much larger, metazoan genomes might remain in the 'Humpty Dumpty' class for a while yet.