Error Correction During Gene Synthesis (NEB #M0689)

In many DIY gene synthesis workflows, users obtain fragments for assembly by purchasing synthesized dsDNA (e.g., gBlocks) or by preparing amplicons of overlapping oligos. Often times, these parts have residual errors arising directly from the oligonucleotides used to generate the fragments. Authenticase will reduce/remove the mismatch/indel (insertion/deletion) regions from amplicons that originate from errors incorporated during the chemical synthesis of oligonucleotides. The following protocol increases the population of correct fragments in the enzyme-corrected DNA pools and subsequently allows DNA polymerase amplification to enrich amplicons with more accuracy and efficiency. The combination of correction and enrichment steps enhances the quality of assembled gene synthesis, thereby producing higher numbers of transformed bacterial colonies with the desired correct DNA sequence.

Process efficiency gains are realized by requiring less colony-picking and sequencing of samples.

Figure: Error Correction During Gene Synthesis Workflow

Note: Authenticase is ONLY USED in the second section of this workflow. We provide the additional info and protocols to enable a more thorough workflow for error correction during DIY gene synthesis. These protocols were optimized and confirmed by NEB^®.

1. Prepare heteroduplex DNA.

The products of the PCR reaction must be denatured and annealed to allow heteroduplex formation between PCR products with and without mutations.

1.1. Clean up dsDNA amplicons using a spin column (e.g., Monarch^® PCR & DNA Cleanup Kit (5 µg) – NEB #T1030) and elute in a small volume (e.g., 12 µl).

1.2. Determine the dsDNA concentration.

1.3. Prepare annealing reaction with DNA concentration around ~40 ng/µl.

1.4. Use a thermocycler to denature and anneal the sample, forming heteroduplex DNA.

2. Treat with Authenticase to digest DNA at mismatches and indels.

2.1. Setup Authenticase cleavage reaction on ice.

2.2. Incubate at 42°C for 60 minutes.

2.3. Add 1.7 µl 150 mM EDTA.

2.4. Heat samples at 95°C for 5 minutes. Tubes can be stored at –20°C until ready to use.

3. Amplify error-depleted population to increase percentage of error-corrected clones.

3.1. Setup reaction for correcting errors of samples from step 2.4.

3.2. Process reaction in a thermocycler to amplify the error-corrected pool.

* Please visit tmcalculator.neb.com to determine correct annealing temperature.

4. Enrich full-size gene of interest.

4.1. At this step, two nearly identical reactions (A&B) are created to amplify and enrich the full-length fragment of interest. The first reaction (Tube A) uses 2 µl of the error corrected pool from step 3.2. as template. The second reaction (Tube B) uses 2 µl from the first reaction (Tube A) as template to ensure an appropriate amount of amplification can be achieved.

4.2. Prepare 2 PCR reactions with 0.5 μM of forward/reverse primers, according to the table below. In the first, add 2 μl of template from step 3.2 to Tube A and mix properly. For the second, transfer 2 μl of Tube A reaction to Tube B. This will create 2 PCR reactions: one with 2 μl (Tube A) and a second with a lower amount (0.16 μl, Tube B) of template from step 3.2. Varying the template to primer ratio can help ensure at least one reaction is productive.

4.3. Amplify full-length fragment of interest in a thermocycler.

* Please visit tmcalculator.neb.com to determine correct annealing temperature.

5. Assemble DNA and transform.

5.1. Check the purity of PCR products from Tubes A and B (we recommend running 10% of the reaction on an agarose gel).

Other methods such as Agilent^® Bioanalyzer^® or TapeStation^® can suffice. Choose the PCR product with higher purity to proceed.

5.2. PCR amplicons are typically cleaned up by spin column prior to quantitation and cloning/DNA Assembly. Reactions can be directly used with reduced efficiency, if desired, although we recommend a cleanup step to ensure accurate quantitation can be performed and to remove potential inhibitors of enzymes used in the next steps of your workflow.

5.3. DNA is ligated or assembled into the destination vector of choice as part of the design strategy. NEBuilder HiFi DNA Assembly or Golden Gate Assembly methods (using NEBridge^® reagents) can be used to assemble DNA.

5.4. Assembled DNA then can be transformed into competent E. coli (e.g., NEB^® 5-alpha or NEB 10-beta) and propagated on rich agar plates with appropriate antibiotic selection.

5.5. Colonies are picked and analyzed for presence of correct clones:

Directly

5.5.1. Directly by colony PCR w/appropriate primers followed by agarose gel electrophoresis to confirm the amplicon, followed by Sanger sequencing (more details in supplemental protocol #2).

OR

Indirectly

5.5.2. By analysis of miniprep plasmid DNA after overnight culture, by restriction enzyme digest or Sanger DNA sequencing.