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Next-Generation DNA Assembly Tools

Emergence of Synthetic Biology Necessitates New Solutions to Generate Molecules

• Lansha Peng
• ,
• Billyana Tsvetanova, Ph.D.
• ,
• Xiquan Liang, Ph.D.
• ,
• Federico Katzen, Ph.D.

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In Vitro Recombineering

Figure 2. In vitro recombineering: (A) Fragments of the indicated size were amplified using PCR SuperMix HiFi or the proofreading Pfx polymerase and recombined into linearized pUC19. End identity was generated by the addition of a 7 to 15 nucleotide tail to the oligonucleotides. (B) Junction editing: Two DNA fragments were recombined at the indicated distances from their corresponding ends. Click Image To Enlarge +

Figure 2. In vitro recombineering: (A) Fragments of the indicated size were amplified using PCR SuperMix HiFi or the proofreading Pfx polymerase and recombined into linearized pUC19. End identity was generated by the addition of a 7 to 15 nucleotide tail to the oligonucleotides. (B) Junction editing: Two DNA fragments were recombined at the indicated distances from their corresponding ends.

In a parallel approach we sought to accomplish DNA fragment assembly in vitro where the assembled molecules are directly selected in E. coli. For this purpose we developed a highly efficient enzymatic mix that promotes homologous recombination of up to four DNA fragments plus a vector with 15 bp end-homology. (Note: we were able to prove assembly of up to seven fragments, but the mix is optimized for up to four fragments plus vector).

DNA molecules (20 to 500 ng per fragment in a 2:1 insert:vector molar ratio) were incubated with the enzyme for 30 minutes  at room temperature, transformed into TOP10 chemically competent cells, and selected on LB agar plates supplemented with the corresponding antibiotic. Cloning efficiencies ranged from 40% to >90% depending on the number and quality of the DNA fragments (Figure 2A).

Recombination can occur not only at the end of the fragments, but it also works at least up to 32 bp away from their ends (Figure 2B). This attribute is useful for generating cloning variants using a single linearized vector.

Figure 3. A novel site-directed mutagenesis strategy: (A) Schematic view of the approach. Template strands are shown in black. Methylated strands are shown as dotted lines. Oligonucleotides and new strands are shown in green and red. (B) Plasmids with frameshift mutations in the lacZa gene were subjected to 12 to 18 PCR amplification cycles using a pair of corrective primers, preceded by a 12-minute step at 37ºC. An aliquot was then subjected to recombination for 10 min at room temp, transformed into DH5a competent cells and plated onto LB agar ampicillin X-gal plates. The mutagenesis efficiency is represented by the ratio blue/total colonies. A 14 kbp plasmid was also subjected to mutagenesis. Ten independent random clones were sequenced, and all of them were correctly mutagenized. Click Image To Enlarge +

Figure 3. A novel site-directed mutagenesis strategy: (A) Schematic view of the approach. Template strands are shown in black. Methylated strands are shown as dotted lines. Oligonucleotides and new strands are shown in green and red. (B) Plasmids with frameshift mutations in the lacZa gene were subjected to 12 to 18 PCR amplification cycles using a pair of corrective primers, preceded by a 12-minute step at 37ºC. An aliquot was then subjected to recombination for 10 min at room temp, transformed into DH5a competent cells and plated onto LB agar ampicillin X-gal plates. The mutagenesis efficiency is represented by the ratio blue/total colonies. A 14 kbp plasmid was also subjected to mutagenesis. Ten independent random clones were sequenced, and all of them were correctly mutagenized.

The enzyme described above can also  be used for recombining and editing the ends of a single DNA molecule, thereby enabling a highly efficient site-directed mutagenesis approach (Figure 3A). Two complementary oligonucleotides with centrally located mutation sites were used.

The DNA methylation and amplification steps were combined into a single reaction. No in vitro digestion or DNA purification was required after methylation or mutagenesis. The system can generate base substitutions, deletions, or insertions of up to 12 nucleotides in plasmids as large as 14 kbp (Figure 3B).