RADOM, an Efficient <i>In Vivo</i> Method for Assembling Designed DNA Fragments up to 10 kb Long in <i>Saccharomyces cerevisiae</i>

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method <i>via</i> homologous recombination in <i>Saccharomyces cerevisiae</i>, which combines assembly <i>in yeasto</i> with blue/white screening in <i>Escherichia coli</i>. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb “minichunks” from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects