Why are the Kinase and Ligase activities not required for the overlapping mutagenic primer strategy? Why is DpnI step still required?

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Wolfgang Schechinger

There are endogenous mechanisms to repair nicks in DNA. DpnI will destroy methylated DNA, i.e. the templates for the mutagenesis.

Weiyan Jia

Wolfgang already answered your questions. I still want to suggest this paper for details and hope it is also helpful for you. I used this protocol for many years, and worked pretty well for most cases.

1.Xia, Y., Chu, W., Qi, Q. & Xun, L. New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis. Nucleic Acids Res 43, gku1189-e12 (2014).