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Editor's Notes

1. dUTPase (dut) and uracil deglycosidase (ung). Both enzymes are part of a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP in the cell. The uracil deglycosidase deficiency prevents the removal of uracil from newly-synthesized DNA. As the double-mutant E. coli replicate the phage DNA, its enzymatic machinery may therefore misincorporates dUTP instead of dTTP, resulting in single stranded DNA which contains some uracils (ssUDNA).
2. Usually the restriction enzymes that cuts at the plasmid and the oligonucleotide are the same, permitting sticky ends of the plasmid and insert to ligate to one another.
3. Site-Directed Mutagenesis by Traditional PCR. Primers incorporating the desired base changes are used in PCR. As the primers are extended, the mutation is created in the resulting amplicon.
4. The overlap-extension method requires four oligonucleotide primers and three separate amplification reactions (34, 35). Two complementary mutagenic primers induce the mutation into the desired sequence of DNA, and two flanking primers amplify the mutant fragment and facilitate cloning of the PCR fragment into a suitable vector. By this approach (Fig. 3), a variety of mutations can be created, such as single base-pair changes, deletions, and insertions. First, two separate PCR reactions are set up in parallel. One reaction has the "sense" mutant primer and an "anti-sense" flanking primer 3′ to the mutation site. The other reaction contains the anti-sense mutant primer and the sense flanking primer 5′ to the mutation site. The two amplified fragments contain mutations at the 5′ or 3′ terminus, respectively. In the second round of amplification, the two fragments from the first round of PCR are purified, then used as templates for amplification using only the flanking primers. After amplification, the mutation is contained within the target DNA segment, which is cloned into appropriate vectors for DNA sequencing and subsequent functional studies. Figure 3. Site-directed mutagenesis by overlap-extension PCR. The two first rounds of PCR produced two overlapping fragments of the original template, both containing the mutation within the overlap region. These two PCR products are annealed and then subjected to a second round of PCR to generate the entire fragment with the mutation. The flanking primers contain the restriction sites for ligating the fragment back into the original vector.
5. The megaprimer method uses only a single mutagenic primer to create mutations in the target template (36-38) (Fig. 4). In the first round of amplification, the wild-type template is amplified using either a sense or anti-sense mutagenic primer and an appropriate flanking primer. The amplified product is then used in a second round of PCR with wild-type template and the other flanking primer to create a fragment of the same length as the original target DNA containing the desired mutation. The key to this method is that the amplified product from the first round of PCR is used as a primer in the second round of PCR. Compared to the four-primer method, this procedure requires only a single mutagenic primer and yields more of the full-length product. This is probably due to the instability of the 10- to 20-basepair overlap between the two mutant templates during the second round of amplification when using the four-primer method. In the megaprimer method, the overlap between the template and mutagenic strands is more extensive. Figure 4. Site-directed mutagenesis by the megaprimer PCR method. The first round of PCR is used to make a fragment of the template DNA containing the desired mutation. This "megaprimer" is then hybridized to the wild-type template DNA, and a second round of PCR is carried out, to generate the entire molecule with the mutation. The flanking primers contain the restriction sites for cloning the fragment back into the vector
6. A third approach, termed inverse PCR, uses only two primers to create the desired mutation (39) (Fig. 5). The key feature of this method is that in making the mutation, the entire vector is amplified. The two primers, one containing the desired mutation, extend on the circular template DNA in opposite directions. Amplification ultimately yields a linear, double-stranded DNA molecule containing the mutation at one end. Following amplification, the ends are ligated, and the resulting circular DNA molecule is transformed into E. coli. There are a number of variations of this method that improve the efficiency of mutagenesis (reviewed in 33). Figure 5. Site-directed mutagenesis by inverse PCR. The vector is cleaved by a restriction enzyme at a single site, close to the site to be mutated. Two primers from the two ends of the linearized molecule, one containing the desired mutation, are used to amplify the linear molecule and produce molecules with the mutation. After ligating the ends to regenerate a circular molecule, the vector is used to transform E. coli.
7. Figure 3. Site-Directed Mutagenesis by Inverse PCR. The primers used are 5’-phosphorylated to allow ligation of the amplicon ends after PCR. A high fidelity DNA polymerase that creates blunt-ended products is used for the PCR to produce a linearized fragment with the desired mutation, which is then recircularized by intramolecular ligation. (A) Deletion: Primers that hybridize to regions on either side of the area to be deleted are used. (B) Substitution: One of the primers contains the desired mutation (blue bubble). (C) Insertion: The primers hybridize to regions on either side of the location of the desired insertion (black, dotted line). One primer contains the additional sequence that will be inserted (blue line).
8. Fig. 1. Schematic diagram of the site-directed mutagenesis method that combines homologous recombination and DpnI digestion of the templates in E. coli BUNDpnI. (A) Protocol for sequence deletion. The target sequence for deletion is denoted in gray, while the flanking regions are denoted in light brown and red. The regions of primers that can hybridize on each strand of the template to gene-specific sequence immediate to the deletion are indicated by arrows, respectively. Fifteen nucleotides immediately 5′ to the region to be deleted (indicated by ab) should be contained in primer F. Step 1: PCR amplification. The desired mutation(s)-containing DNA molecules with a homologous region at the terminal ends were created by PCR. Step 2: Transformation. The mutated plasmid was efficiently generated on direct transformation of the amplified PCR products into E. coli BUNDpnI. (B) Sequence insertion. As for sequence insertion, to simplify the figure, only the initial primer binding sites and the final inserted products are depicted. The insertion is denoted by a red and green rectangle. The gene-specific regions of primers that can hybridize on each strand of the template are located immediate to the site of the insertion, respectively. The sequence to be inserted is incorporated at the 5′ end of primer F. Primer R contains a 5′ tail complementary to the 15 outermost nucleotides of primer F (indicated by a red line). The final product with the inserted sequence denoted by the red and green rectangles was obtained after transformation. (C) Point mutations. Only the initial primer binding sites are shown. The region to be mutated is represented by a red rectangle, with the target site indicated by an asterisk. The desired nucleotides (indicated by black square dots) to be introduced into the target sequence are incorporated into the overlapping region of primers. The products carrying the designed mutations are generated by PCR amplification as described for sequence deletion.