*2.12. Harvest of the Total DNA and Amplification of the Target DNA Sequence*

The total DNA was harvested from the cell culture including the cell pellet and supernatant using the Blood and Tissue DNeasy kit (Qiagen, Germantown, MD, USA) and quantified by UV260 with a spectrophotometer. We amplified the specific product using MCMVM80/80.5 primers and the total DNA as a template by a polymerase chain reaction (PCR), under the conditions of 300 nM for each primer and 1× Hotstar Taq master mix (Qiagen, USA) in a 50 μL mixture. The thermal cycling conditions were 95 ◦C for 15 min followed by 35 cycles of 94 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 1 min, and 72 ◦C for 10 min.

#### *2.13. Surveyor Nuclease Mutation Detection Assay*

A Surveyor nuclease mutation detection kit (Surveyor nuclease and G + C control included) was obtained from IDT Integrated Technologies, USA [23].

We amplified wild-type (reference) and mutant (test) total DNA by PCR using MCMV M80/80.5 primers. We mixed equal amounts of reference and test PCR products. We incubated the mixture at 95 ◦C for 5 min in a beaker filled with 800 mL of water. We then allowed the mixture to denature in order to rehybridize, by heating and cooling it to form heteroduplexes and homoduplexes (finally leaving the water at <30 ◦C). We treated the annealed mixture with the Surveyor nuclease and incubated at 42 ◦C for 1 h. The reference PCR product was treated alone as a negative control. DNA fragments were separated by 2% agarose gel electrophoresis [23].

The cleavage efficiency of PCR products was calculated by scanning the signal strength of DNA bands on the UV illuminator. They were indicated by the percentage (%) of extra bands divided by the total bands (major bands + extra bands) in signal strength.
