Assembling large DNA segments in yeast

Héloïse Muller, Narayana Annaluru, Joy Wu Schwerzmann, Sarah M. Richardson, Jessica S. Dymond, Eric M. Cooper, Joel S. Bader, Jef D. Boeke, Srinivasan Chandrasegaran

    Research output: Chapter in Book/Report/Conference proceedingChapter

    Abstract

    As described in a different chapter in this volume, the uracil-specific excision reaction (USER) fusion method can be used to assemble multiple small DNA fragments (∼0.75-kb size) into larger 3-kb DNA segments both in vitro and in vivo (in Escherichia coli). However, in order to assemble an entire synthetic yeast genome (Sc2.0 project), we need to be able to assemble these 3-kb pieces into larger DNA segments or chromosome-sized fragments. This assembly into larger DNA segments is carried out in vivo, using homologous recombination in yeast. We have successfully used this approach to assemble a 40-kb chromosome piece in the yeast Saccharomyces cerevisiae. A lithium acetate (LiOAc) protocol using equimolar amount of overlapping smaller fragments was employed to transform yeast. In this chapter, we describe the assembly of 3-kb fragments with an overlap of one building block (∼750 base pairs) into a 40-kb DNA piece.

    Original languageEnglish (US)
    Title of host publicationGene Synthesis
    Subtitle of host publicationMethods and Protocols
    EditorsJean Peccoud
    Pages133-150
    Number of pages18
    DOIs
    StatePublished - 2012

    Publication series

    NameMethods in Molecular Biology
    Volume852
    ISSN (Print)1064-3745

    Keywords

    • DNA assembly
    • Large DNA fragments
    • Synthetic yeast
    • USER fusion

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics

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