Hi Chunyu Hu ,

It depends on how far those mutations are from each other. There are many different methods for mutagenesis. You may try classical site-directed mutagenesis but you will probably need very long primers purified by PAGE and it can be quite expensive and tedious.

If you find two restriction target sites that flank the sequence that you want to mutate, you can cut the plasmid and use as insert two pre-annealed complementary primers with the mutations and the proper overhang at each end (don't dephosphorylate your plasmid or ask for phosphorylated primers in this case).

Another possibility is to use splicing overhang-extension PCR to "sew" several fragments.

Yes, it is possible to perform mutagenesis to introduce 9 mismatches in one round of PCR. There are several protocols you can learn from:

1. One-Step Overlap Extension PCR (OOE-PCR): This method allows efficient site-directed mutagenesis in a single reaction without the need for purification of intermediate products. It is particularly useful for introducing multiple mutations simultaneously [1].

2. Combined Overlap Extension PCR (COE-PCR): This method combines the strengths of overlap extension PCR (OE-PCR) and asymmetrical overlap extension (AOE-PCR), enabling the mutation of up to 6 base pairs at a time in a single experiment. It has been successfully used to mutate between two to six base pairs, changing up to four adjacent codons in one experiment [2].

3. Rapid One-Tube PCR-Based Mutagenesis: This technique employs Pfu DNA polymerase for high fidelity and does not require purification of intermediate products. The method can be completed in less than 4.5 hours with high efficiency, making it suitable for introducing multiple mutations [3].

These methods provide efficient ways to introduce multiple mutations, including up to 9 mismatches, in a single round of PCR.

References

[1] Urban, A., Neukirchen, S., & Jaeger, K. (1997). A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.. Nucleic acids research, 25 11, 2227-8 .

[2] Hussain, H., & Chong, N. F. M. (2016). Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis. BioMed Research International, 2016.

[3] Picard, V., Ersdal-Badju, E., Lu, A., & Bock, S. C. (1994). A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic acids research, 22 13, 2587-91 .