Complementary oligonucleotide primers of about 30 nucleotide long with the desired point mutation in the middle of primer should be designed. Two sets of PCR amplification with an outer primer & a mutation primer would yield two amplicons in the first round of PCR. A second round of PCR with EQUIMOLAR concentration of first round amplicons with outer extension primers will result in full-length amplicons with the desired point mutation. The annealing temperatures for amplification would be set according to the Tm of the primers. Hope it helps.

best

Sandeep

Two first round amplicons, you can prepare using the method discussed above by Sandeep Upadhyay. If both the amplicons have same intensity, you can go ahead with 1:1 ratio of them for extension PCR. Annealing time during PCR should be more than that you set for first round PCR. Also annealing temprature should be according to the Tm of restriction primers. Sometimes annealing temprature also depends upon the type of polymerase you are using; like for KapaHifi, vent polymerase, it should be -3 of the Tm of the restriction primer while for Phusion poly. it should be +3 of Tm of restriction primers.

Thank you...