Figure 1. Mutagenic oligonucleotide design. The blue box represents the target gene. The short arrowhead line represents the oligonucleotide. The cross represents desired mutation. P, phosphate group at 5¢-end of DNA, m, methyl groups. (a, b) Single or nearby multiple mutations can be designed into the middle area of one or a pair of complementary mutagenic oligos, which will lead to the synthesis of the whole plasmid. (c) Mutations at the terminal regions of a gene can be designed into a pair of mutagenic oligos, which will lead to the synthesis of the target gene. (d) Scattered multiple mutations can be designed into a pair of outside mutagenic oligos (a and d) and a pair of middle mutagenic oligos (b and c). Mutations in b and c oligos sit in the flanking areas that cannot anneal with the template but are complementary to each other. Polymerase chain reaction (PCR) products from oligos a and c and oligos b and d can anneal to each other and be elongated to form dsDNA. Thus, the mutated gene can be generated by a final PCR with oligos a and d. (e) Multiple mutations separated by long distance can be made in one PCR, using one oligo per mutation. All the oligos anneal to the same strand of DNA. The gaps between the oligos are filled and ligated to generate ssDNA. The ssDNA is then converted into dsDNA in vivo in the host cell (see Figure 10 for a more detailed description).

Figure 2. In vitro mutagenesis using dut^– ung^– genetic selection method.

Figure 3. Schematic diagram of in vitro mutagenesis procedure using the pALTER^®-1 vector as an example (Promega protocol). Target gene was cloned in a plasmid with both tetracycline-resistant gene and point-mutated ampicillin-resistant gene (Amp^S). Bacterial cells transformed with the plasmid are tetracycline resistant, but ampicillin sensitive. Mutagenic oligo is made containing the desired mutation for the target gene. Meanwhile, Amp^r oligo is made containing the mutation, which can repair the point-mutated ampicillin-resistant gene back to its wild-type coding sequence. Alkaline-denatured plasmid, ssDNA can anneal to both mutagenic and Amp^r oligo. The mutant DNA strand can be synthesized with T4 DNA polymerase and T4 DNA ligase. The generated plasmid was first transformed to mismatch-repair-deficient cell ES1301 mutS allowing the formation of double-stranded mutant plasmid that can be selected with ampicillin plates. Purified plasmid DNA from surviving ES1301 mutS was then transformed to JM109 grown on ampicillin plates to select and amplify mutant plasmid.

Figure 4. Thionucleotide selection-based mutagenesis. The blue box represents the target gene. The short arrowhead line represents the mutagenic oligo. The peak arch represents desired mutation. ‘A’ represents restriction endonuclease and its site. The incorporation of a certain type of deoxynucleotide triphosphate S (dNTPS) can prevent A restriction, resulting in a nick in the DNA strand without thionucleotide incorporation.

Figure 5. Unique site elimination-based mutagenesis. The blue box represents the target gene. The short arrowhead line represents the mutagenic oligo (with peak arch) or selection oligo (with square). The peak arch in the blue box represents desired mutation. ‘A’ represents unique restriction endonuclease, and the semicircle on the plasmid represents the sequence for unique restriction enzyme A.

Figure 6. Site-directed mutagenesis method based on the QuikChange method. Two complementary primers with the desired mutation are used in a PCR to synthesize mutated DNA. The parental strands, methylated before PCR by dam⁺ E. coli host, is digested by DpnI (restriction endonuclease cut at G^meA TC site) and the nicked vector with the mutation was transformed into XLI Blue. The mutation is verified by sequencing.

Figure 7. GeneTailor Site-Directed mutagenesis system. The short arrowhead line represents oligonucleotide, m, methyl group. The DNA is methylated in vitro by DNA methylase. In the following mutagenesis reaction, the DNA is amplified with two overlapping oligonucleotides, one that contains the mutation. The DNA is transformed into DH5™-T1^R cells. These cells contain McrBC endonuclease, which digests the methylated parental DNA.

Figure 8. Outline of the mutagenesis strategy at multiple sites. The short arrowed line represents oligonucleotide, m, methyl group; P, phosphate group at 5¢-end of DNA. All the oligos are designed to anneal to the same strand of DNA, one oligonucleotide per mutation. The polymerase extends the primers without strand displacement, and the nicks created are sealed by the components of the enzyme blend. The methylated parental DNA is restricted by DpnI (restriction endonuclease cut at G^meA TC site). The ss-mutated-DNA is converted to dsDNA in vivo in XL-10-Gold^® cells.

Figure 9. Overview of the inverse PCR-based site-directed mutagenesis protocol (Fisher and Pei, (1997)), ExSite. This method requires phosphorylated primer(s). It permits insertions and deletions. DpnI, restriction endonuclease cut at G^meATC site; m, methyl group; P, phosphate group at 5¢-end of DNA.