Gene Synthesis using Gibson Mix and Error Correction Enzymes

Synthetic gene products were assembled from a plurality of oligonucleotides with overlapping sequences at their terminal ends using Gibson Assembly™ (Synthetic Genomics, Inc., San Diego, Calif.) as previously described (see, e.g., US Patent Application No. 2010/0035768). Genes included representative hemagglutinin (HA) genes and representative neuraminidase (NA) genes.
Reagents 5× Isothermal (ISO) Buffer:
[0104]
The 5×ISO buffer contains 25% PEG-8000, 500 mM Tris-HCl pH 8.0, 50 mM MgCl2, 50 mM DTT, 1 mM each of the 4 dNTPs, and 5 mM NAD.
[0105]
Six ml of this buffer can be prepared by combining the following:

        3 ml of 1 M Tris-HCl pH 8.0
        300 μl of 1 M MgCl2
        600 μl of 10 mM dNTPs
        300 μl of 1 M DTT (1.54 g dissolved in dH2O up to 10 ml)
        1.5 g PEG-8000
        300 μl of 100 mM NAD (Sigma; 0.66 g dissolved in dH2O up to 10 ml; resuspend by heating at 50° C. followed by continuous vortexing)
        Add water to 6 ml, aliquot 1 ml and store at −80° C.

2× Assembly Master Mix:
[0113]
The 2× Assembly Master Mix contains the ISO reaction buffer and the enzymatic activities required for assembly of the component oligonucleotides into the gene products: 5× isothermal (ISO) reaction buffer) T5 exonuclease (Epicentre), PHUSION® DNA polymerase (Finnzymes Oy, Vantaa, Finland) and Taq DNA ligase.
[0114]
800 μl of the 2× assembly master mix, sufficient for 80 reactions, can be prepared by combining the following:
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320 μl 5×ISO buffer as prepared above
[0116]
6.4 μl of 1 U/μl T5 exo (diluted 1:10 from enzyme stock in 1×T5 exo buffer)
[0117]
20 μl of 2 U/μl PHUSION®polymerase (Finnzymes Oy, Vantaa, Finland)
[0118]
80 μl of 40 U/μl Taq ligase
[0119]
374 μl dH2O
[0120]
Mix well and store at −20° C., or on ice if to be used immediately.
[0121]
The assembly mixture can be stored at −20° C. for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. The mixture is ideal for the assembly of DNA molecules with 20-150 bp overlaps.
Oligonucleotides:
[0122]
Standard oligonucleotides were purchased at a concentration of 10,000 nM each. The entire HA gene sequence was covered using 52 oligonucleotides (oligos), and the entire NA gene sequence was covered using 44 NA oligonucleotides.
Assembly of Genes
[0123]
For the assembly reactions 10 μl of each oligo was pooled, for a concentration of 192 nM per oligo (10,000 nM/52) for HA and 227 nM per oligo (10,000 nM/44) for NA. Oligo lengths were on average 60 bases with 30 bp overlaps.
[0124]
Once pooled, 10 μl of this oligo mix was added to 10 μl of the 2× assembly master mix prepared as above. Reactions were incubated for 50° C. for 1 hour. Following assembly reactions, the gene products were amplified by PCR as follows:
[0125]
5 μl assembly reaction
[0126]
20 μl 5× PHUSION® HF Buffer (Finnzymes Oy, Vantaa, Finland)
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2 μl 10 mM dNTPs
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71 μl water
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1 μl Hot Start PHUSION® Polymerase (Finnzymes Oy, Vantaa, Finland)
[0130]
0.5 μl 100 uM RC-Univ-PKS10-F primer (universal forward primer for cloning vector)
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0.5 μl 100 uM RC-Univ-PKS10-R primer (universal reverse primer for cloning vector)
[0132]
Cycling reaction was as follows:
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98° C. for 1 minute,
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98° C. for 10 seconds, 60° C. for 30 seconds, 72° C. for 1.5 minutes,
[0135]
Repeated for 24 additional cycles, 72° C. for 5 minutes, and then kept at 4° C.

Error Correction Two-Step Reactions

In the two-step reactions, the endonuclease enzyme reaction was performed first, followed by the exonuclease enzyme reaction. Following PCR of the assembled gene product as indicated in Example 1, the following reactions were performed. [0157] SURVEYOR® nuclease/Exonuclease III

       8 μl of the PCR product was denatured and annealed as follows:
      98° C. for 2 minutes, slow cool to 85° C. at 2° C./second, hold at 85° C. for 2 minutes, slow cool to 25° C. at 0.1° C./second, hold at 25° C. for 2 minutes, and then hold at 10° C.
      2 μA SURVEYOR® nuclease (Transgenomic, Inc., Omaha, Nebr.) (a mismatch endonuclease derived from the celery plant that cleaves all types of mismatches) was added and incubated at 42° C. for 1 hour.
      1 μl Exonuclease III (diluted 1 to 4000 in 1×HF Buffer) was added and incubated at 37° C. for 1 hour.
      2 μl of this reaction mix was then amplified by PCR as described above.
      Optionally, steps 1-3 were repeated to increase fidelity (to further reduce errors in the final gene product)

from 46.4% to 80% with an error rate improvement of 1/1850 bp to 1/5314 bp using the two-step error correction method combining an endonuclease enzyme with an exonuclease enzyme

Error Correction One-Step Reactions

[0173] In the one-step reactions, the endonuclease enzyme reaction was performed simultaneously (in the same reaction mixture at the same time) with the exonuclease enzyme reaction. Following PCR of the assembled gene product as indicated in Example 1, the following reactions were performed.

       8 μl of the PCR product was denatured and annealed as follows:
      98° C. for 2 minutes, slow cool to 85° C. at 2° C./second, hold at 85° C. for 2 minutes, slow cool to 25° C. at 0.1° C./second, hold at 25° C. for 2 minutes, and then hold at 10° C.
      2 μl SURVEYOR® nuclease (Transgenomic, Inc., Omaha, Nebr.) and 1 μl Exonuclease III, (diluted 1 to 4000 in 1×HF Buffer) were added and incubated at 42° C. for 1 hour.
      2 μl of this reaction mix was then then amplified by PCR as described above.

[0178] Optionally, steps 1 and 2 were repeated to increase fidelity.

One-step error correction methods can be readily performed at 42° C.