The Seven Stones


A few weeks ago, Jason Kelly explained in his post how Itaya and colleagues (2007) assembled the complete 135 kb rice chloroplast circular genome starting from a collection of 5-6 kb fragments and using sequential in vivo homologous recombination in Bacillus subtilis. Now, Hamilton Smith, Craig Venter and colleagues have achieved the assembly of a complete 583 kb Mycoplasma genitalium genome (“JCVI-1.0”, Gibson et al, 2008). The starting fragments were of similar length, 4-5 kb fragments with 80-360 kb overlaps, albeit synthesized chemically rather than by PCR. In contrast to Itaya et al, Ham Smith’s team used in vitro recombination (using T4 pol digestion/annealing/Taq pol repair and ligation) in a 3 step hierarchical assembly process and completed the fourth step, the assembly of 4 quarter genomes, using in vivo homologous recombination in yeast (TAR cloning, Larionov et al, 1996). 


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