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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