Site-directed mutagenesis in very large cDNA

Adapted from Zhang K, Yin X, Shi K, et al. A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation.

Sci Rep. 2021;11(1):10454. doi:10.1038/s41598-021-89884-z

Reagents

• 2 × Phanta Flash Master Mix (Vazyme P510)
• ClonExpress II One Step Cloning Kit (Vazyme C112)

Primer Design

Design a pair of mutagenesis primers (MFP and MRP) and a pair of mutation-assisting primers (MAFP and MARP).

• The distance between two pairs of primers may vary from 600bp to 15Kb
• The length of each primer is about 20bp
• For each pair of primers, the overlap region is about 15bp
• The mutation should fall in the middle of the overlap region in MFP and MRP (around 6bp from the 5’ end)
• The GC content for each primer is between 45% and 65% and avoid over 4 consecutive G or other bases

PCR reaction

1. Prepare 2 PCR reactions:
   1. MAFP and MRP
   2. MARP and MFP
2. For each 40µl PCR reaction, add
   1. 0.25ng input plasmid
   2. 1µl 25mM forward primer
   3. 1µl 25mM reverse primer
   4. 20µl 2x Phanta Flash master mix
   5. Add H₂O up to 40µl
3. Run PCR reactions with the cycling conditions
   1. 98°C, 30s
   2. 32 cycles of
      1. 98°C, 10s
      2. 56°C (or Tm of primers), 5s
      3. 72°C, 5s/kb if <10kb, 10s/kb if >10kb
   3. 72°C 1min
   4. 4°C hold

Gel electrophoresis and PCR cleanup

4. Use 5µl PCR products for agarose gel electrophoresis
5. After validating the PCR products, cleanup using NucleoSpin kit
   1. Mix 35µl PCR product with 70µl buffer NTI
   2. Add to clean-up column and centrifuge at 11,000 xg for 30s
   3. Add 700µl buffer NT3 and centrifuge at 11,000 xg for 30s
   4. Repeat step 3
   5. Centrifuge at 11,000 xg for 1 min to dry silica membrane
   6. Place column in a fresh eppendorf and add 15µl buffer NE
   7. Incubate at RT for 1 min and centrifuge at 11,000 xg for 1 min 
6. Nanodrop to check concentration

Recombinational ligation

7. Prepare 20µl recombinational ligation reaction
   1. Mix equal moles of PCR fragments (use around 10ng/kb fragment)
   2. 1-2µl Exnase II
   3. 4µl 5x CE II buffer
   4. Add H₂O up to 20µl
8. For a positive control, use 0.5µl of the pUC19 plasmid and insert included in the kit
9. For negative controls, use the PCR fragment containing the antibiotic-resistance region, without adding Exnase II
10. Incubate at 37°C for 30min and keep on ice immediately
11. Use 3µl ligation sample for transformation

Validation

12. Run 5µl Miniprep plasmid on an agarose gel to validate ligation products
13. Validate mutations by Sanger sequencing