Skip to main content

Assembling Large DNA Segments in Yeast

  • Protocol
  • First Online:
Gene Synthesis


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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others


  1. Dymond, JS, Scheifele, L., Richardson, S, Lee, P, Chandrasegaran, S, Bader, JS, Boeke, JD (2009) Teaching synthetic biology, bioinformatics and engineering to undergraduates: the interdisciplinary Build-a-Genome course. Genetics 18:1321.

    Google Scholar 

  2. Cooper E, Book chapter 20. THE BUILD A GENOME CLASS.

    Google Scholar 

  3. Chandrasegaran S, Book chapter 7. USER FUSION.

    Google Scholar 

  4. Gibson, DG, Benders, GA, Andrews-Pfannkoch, C, Denisova, EA, Baden-Tillson, H, Zaveri, J, Stockwell, TB, Brownley, A, Thomas, DW, Algire, MA, Merryman, C, Young, L, Noskov, VN, Glass, JI, Venter, JC, Hutchison, CA 3rd, Smith, HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:121520.

    Article  PubMed  CAS  Google Scholar 

  5. Gibson, DG, Benders, GA, Axelrod, KC, Zaveri, J, Algire, MA, Moodie, M, Montague, MG, Venter, JC, Smith, HO, Hutchison, CA 3rd (2008) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci USA 105:204049.

    Article  PubMed  CAS  Google Scholar 


    Google Scholar 

  7. Richardson, SM, Wheelan, SJ, Yarrington, RM, Boeke, JD (2006) GeneDesign: rapid, automated design of multikilobase synthetic genes. Genome Res 16:5506.

    Article  PubMed  CAS  Google Scholar 

  8. Richardson, SM, Nunley, PW, Yarrington, RM, Boeke, JD, Bader, JS (2010) GeneDesign 3.0 is an updated synthetic biology toolkit. Nucleic Acids Res 38:26036.

    Article  PubMed  CAS  Google Scholar 

  9. Richardson S, Book chapter 18 GENE DESIGN.

    Google Scholar 

  10. Gietz, RD, Schiestl, RH, Willems, AR, Woods, RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:35560.

    Article  PubMed  CAS  Google Scholar 

Download references


This work was supported by grants from National Science Foundation (MCB0718846) to JDB, JSB, and SC; from Microsoft to JSB and JDB; and from National Institutes of Health (GM077291) to SC. HM was a recipient of a fellowship from the Fondation pour la Recherche Medicale (FRM).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Srinivasan Chandrasegaran .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Sceince+Business Media, LLC

About this protocol

Cite this protocol

Muller, H. et al. (2012). Assembling Large DNA Segments in Yeast. In: Peccoud, J. (eds) Gene Synthesis. Methods in Molecular Biology, vol 852. Humana Press.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-563-3

  • Online ISBN: 978-1-61779-564-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics