RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in saccharomyces cerevisiae

Qiuhui Lin, Bin Jia, Leslie A. Mitchell, Jingchuan Luo, Kun Yang, Karen I. Zeller, Wenqian Zhang, Zhuwei Xu, Giovanni Stracquadanio, Joel S. Bader, Jef D. Boeke, Ying Jin Yuan

    Research output: Contribution to journalArticlepeer-review

    Abstract

    We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. 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.

    Original languageEnglish (US)
    Pages (from-to)213-220
    Number of pages8
    JournalACS Synthetic Biology
    Volume4
    Issue number3
    DOIs
    StatePublished - Mar 20 2015

    Keywords

    • Build-A-Genome
    • Sc2.0
    • in vivo assembly
    • synthetic biology
    • synthetic yeast genome

    ASJC Scopus subject areas

    • Biomedical Engineering
    • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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