This technique is a modification of the protocol of commercial site-directed mutagenesis kits and relies on a simultaneous annealing of specific mutagenic oligonucleotides on different sites of the template, to obtain in a single reaction single and/or multiple mutations (1,2). We have used this strategy to introduce two amino acid substitutions, S46A and S64A, in the human Tumor Protein Translationally-Controlled 1 gene (TPT1)[EMBL:BC040008.1].

The two couples of complementary oligonucleotides used to mutagenize cDNA, were obtained from web-based program PrimerX (bioinformatics.org/primerx).
Each pair of oligonucleotides is about 40 bp long and contains the specific substitution in the middle of the sequence.

The Stratagene Quickchange site-directed mutagenesis protocol provides a description of the method and is available as a PDF file at: (chem.agilent.com/Library/usermanuals/Public/210518.pdf).

Procedures

PCR assembly

• DNA template plasmid 50 ng
• 10X Phusion DNA polymerase buffer 5 µl
• 120 ng of each oligonucleotide primer
• 1 µl of 10 mM dNTP mix
• 1 µl of Phusion DNA polymerase (M-MEDICAL)
• Fill with dd H₂O to 50 µl

PCR conditions

95°, 2’(1 cycle); 18 cycles: (95°, 20”; 60°,10”; 68°,2 minutes/kb of plasmid length) ( 68°, 5’), one final cycle.

After PCR amplification, add 1 µl of DpnI restriction enzyme to half of the reaction and incubate for 2 hr at 37°. Use 12 µl of the digested product to transform DH5α chemically competent E.coli.(3). Sequence 5-10 of the resulting colonies.

Results

By sequencing 5 colonies of 57 obtained, 40% of colonies were double mutated, 60% were mutated in only one site (among these 20% were mutated also in unspecific sites).

References

1. Liu H, Naismith JH. (2008) - BMC Biotechnology 8(1):91. PMID:19055817
2. Kirsch RD and Joly E. (1998) - Nucleic Acids Research 26:1848-1850. PMID:9512562
3. Chung,C.T and Miller,R.H. (1988) - Nucleic Acids Res. 16(8),3580. PMID:3287331