by Karmella Haynes and Cameron Gardner, 2015
Based on de Kok, S., Stanton, L. H., Slaby, T., Durot, M., Holmes, V. F., Patel, K. G., et al. (2014). Rapid and reliable DNA assembly via ligase cycling reaction. ACS synthetic biology, 3(2), 97–106. doi:10.1021/sb4001992
Principle: The DNA parts to be assembled are blunt-ended PCR products (generated by Phusion polymerase). 5' phosphates are added by polynucleotide kinase or 5'-phos primers. After (initial) denaturation at high temperature, the upper (or lower) strands of neighboring DNA parts anneal with non-phosphorylated Bridge Oligos, complementary single-stranded DNAs that span the junctions between adjacent DNA parts. Thermostable ligase joins the DNA backbones via a phosphodiester bond without introducing any scar sequences. In subsequent denaturation−annealing−ligation temperature cycles, the Bridging Oligos are replaced by complementary strands from the DNA parts and that backbone is sealed. By applying multiple temperature cycles, many DNA parts can be assembled into complex DNA constructs.
Part 1: Design the Assembled Plasmid, bridge oligos, and primers in silico
- Use a DNA editing program (e.g., Benchling) to create a file in which the DNA fragments and vector of interest are assembled into the desired construct.
- Locate the first "junction": the boundary between the vector (left) and the first DNA fragment (right).
- Select ~20 bp starting at the junction going leftward into the vector that has a melting temperature (Tm) of 60°C. This will be the left half of the bridge oligo for this junction.
- Select ~20 bp starting at the junction going rightward into the DNA fragment that has a melting temperature (Tm) of 60°C. This will be the right half of the bridge oligo for this junction.
- Annotate the Bridge Oligo as the complementary (minus strand) sequence that includes both the left and right half you identified in steps 3 and 4.
- Repeat this step for all remaining junctions.
- Example 1: If your assembly has one insert and one vector, you will have two junctions and will design two Bridge Oligos
- Example 2: If your assembly has two inserts and one vector, you will have three junctions and will design three Bridge Oligos
Part 2: Prep the Bridge Oligos
- Note: Final conc. in LCR rxn. is 30 nM each
- Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add
- Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL
- Use 3.0 μL of oligo working sln. per 30 μL LCR reaction
- EDIT: Instructions above would make a 3 µM working solution of bridge oligo and a final concentration of 300 nM. Instead, to make a true 300 nM working solution, either start with 10 µM stocks or dilute 0.3 µL of 100 µM stock into 97 µL H2O. - Ben
Part 2: Prep the DNA fragments (PCR & PNK)
- Note: Final conc. in LCR rxn is 3 nM each
- Amplify the fragment(s) of interest in 50 μL Phusion polymerase PCR reactions. Use Phusion polymerase to generates fragments with blunt ends (others produce T/A overhangs).
- Purify the product with a kit of choice (e.g. Sigma PCR clean-up)
- Measure the ng/μL of the purified sample.
- Dilute the purified dsDNA to 30 fmol/μL (30 nM)
- The volume of purified DNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * 30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng * 50 μL final volume.
- Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
- Use 2.0 μL of each diluted dsDNA per 10 μL PNK reaction
- Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)
- OPTIONAL - Digest the template DNA: Include 1 μL FastDigest DpnI in the PNK reaction. DpnI will cut only methylated DNA that came from a bacterial cell (plasmid template) and not the synthetic PCR product.
|Clean PCR or dsDNA||up to 8.5 μL|
|10x T4 Ligation buf (NEB)||1.0|
|T4 PNK (NEB)||0.5|
- Incubate at 37°C/ 30 min.
- Heat-inactivate PNK at 65°C/ 20 min.
Part 3: Perform LCR
- Add components to the PNK DNA reaction as described in the table below
- Set up the reactions in PCR-sized tubes
|PNK DNA||10 μL|
|Oligo Bridge||3.0 (each)|
|10X Ampligase Buffer||3.0|
- Thermal cycler program
Part 4: Perform Transformation
- Use a method of choice to transform 20 μL into 50 μL competent bacteria
- Cameron used ~1μL of ~24 ng/μL insert (restriction digest)
- This measured ng/μL is too low for the dsDNA dilution step (x = ~90, which is greater than 50)
- Cameron used ~36.93 fmol in a 25 μL LCR = 1.47 fmol/μL (1.47 nM), whereas the recommended amount is 3.0 fmol/μL (3 nM)
- Also appears that Tm's were not optimized to 60°C for each half of the bridge oligo