(a) Site-directed mutagenesis is accomplished by using mutagenic primers (b and c) and flanking primers (a and d) to generate intermediate PCR products AB and CD that are overlapping fragments of the entire product AD. Products AB and CD are denatured when used as template DNA for the second PCR; strands of each product hybridize at their overlapping, complementary regions that also contain the desired mutation (indicated by the cross). Amplification of product AD in PCR #2 is driven by primers a and d. Final product AD can be inserted into an expression vector (gray circle) to generate larger quantities of DNA, which should also be sequenced to ensure the presence of the desired mutation.
(b) Chimeric gene products can be generated by two PCRs, as in a, except that internal primers b and c are not mutagenic. Instead, because the goal here is to splice together two different gene segments, primers b and c generate overlapping sequences by including nucleotides that span the junction of segments AB (solid line) and CD (dashed line). The second PCR generates the hybrid gene product AD that is then ready to insert into a vector (gray circle) for larger-scale production and verification of the accurate joining of gene segments AB and CD.