(A) 5′ add-on mutagenesis. Primers can be modified at the 5′ end to introduce, for example, a labeled group (Figure 10.24), a sequence containing a suitable restriction site (Figure 20.12) or a phage promoter to drive gene expression. (B) Site-specific mutagenesis. The mutagenesis shown can result in an amplified product with a specific pre-determined mutation located in a central segment. PCR reactions A and B are envisaged as amplifying overlapping segments of DNA containing an introduced mutation (by deliberate base mismatching using a mutant primer - 1M or 2M). After the two products are combined, denatured and allowed to reanneal, the DNA polymerase can extend the 3′ end of heteroduplexes with recessed 3′ ends. Thereafter, a full length product with the introduced mutation in a central segment can be amplified by using the outer primers 1 and 2 only.