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Stanford Genome Technology Center


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DNA Assembly by Yeast Homologous Recombination

Method Overview

Recent developments in DNA sequencing and synthesis technologies have made it much easier to obtain any customized pieces of DNA. While the length of oligonucleotides that can be commercially purchased have become increasingly longer, broadly applicable and highly efficient DNA assembly methods are still critical for constructing and studying genetic or metabolic pathways.

At the SGTC, we have adopted one of the recently developed protocols: DNA assembly by yeast homologous recombination, to construct larger pieces of genetic fragments from oligonucleotides or smaller DNA fragments. Such method involves an in vitro Overlap-Extension PCR (OE-PCR) reaction followed by an in vivo S. cerevisiae homologous recombination, all based on sequence homology. This method has been used for a 42 kb assembly in one reaction, and has the potential to accommodate longer genetic fragments. Final constructs can be in plasmid format or fragments can be integrated into the genome. All the sequence homology can be constructed via PCR reactions and no scars are left between the fragments joined in this process.

Materials:

Q5® Hot Start High-Fidelity 2X Master Mix (NEB, Cat. #M0494S)
Genetic fragments and PCR Oligonucleotide Primers
Reagents for high efficiency Lithium Acetate (Li/Ac) yeast transformation
Reagents for PCR purification and DNA gel purification (Qiagen or other suitable vendors)

Protocol

OE-PCR (Adapted from openwetware.org/wiki/PCR_Overlap_Extension)

  1. Design Primers:
    1. These primers are like bridges between the two parts you want to assemble together.
    2. You will order two primers which are complements of one another.
    3. These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
    4. The "end primers" will not have any complements and will likely only have restriction sites.

  2. "Extension PCR" PCR amplify the necessary fragments separately

  3. Clean up the product using a DNA column.

  4. "Overlap PCR" Use cleaned up fragments as template in a PCR reaction:
    1. About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
    2. Do not add any primers; the templates will prime each-other.
    3. Run 10 PCR cycles without primers.
    4. Use an annealing temp of 58°C.

    5. An example cycling condition is shown below*:

      Thermal cycle

      98C, 30 sec.

      10 cycles @

      |98C, 10 sec
      |58C, 30 sec
      |72C, 30 sec

      72C, 2 min.
      4C, hold


  5. "Purification PCR" Add end primers to the Overlap PCR reaction:
    1. Continue cycling for another 35 rounds.
    2. Use an annealing temp of 60°C.

    3. An example cycling condition is shown below*:

      Thermal cycle

      98C, 30 sec.

      35 cycles @

      |98C, 10 sec
      |60C, 30 sec
      |72C, 30 sec

      72C, 2 min.
      4C, hold


    4. Gel extract the correct size fragment.

    *For all the aforementioned PCR reactions, an example set-up with Q5 enzyme is available at NEB website: https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491

    Yeast Homologous Recombination:

    1. Prepare 500 ng of DNA for each fragments assembled. Prepare 150 ng of DNA for the cut plasmid backbone.

    2. Transform the mixture using a high efficiently Li/Ac yeast transformation protocol.

    3. Screen transformants for correctly assembled clones.

    References

    1. Shao Z, Zhao H, and Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res, 2009. 37: p.e16. PMCID: PMC2632897
    2. Horton RM, Cai Z, Ho SM, Pease LR. Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction. Biotechniques, 2013. 54: p. 129-133. PMID: 23599925
    3. Chao R, Yuan Y, Zhao H. Recent advances in DNA assembly technologies. FEMS Yeast Res, 2014 . p1567-1364. PMCID: PMC4257898

 



Inquiries can be addressed to Maureen Hillenmeyer (maureenh at stanford.edu) and Angela Chu (amchu at stanford.edu)
Stanford Genome Technology Center