The next step is to design the primers, which may require use of
both methods (Fig. 1), depending on the availability of suitable
restriction sites in both the vector and the insert. With conventional
cloning, such sites are often not present and need to be
introduced by polymerase chain reaction (PCR), or existing restriction
sites need to be removed by site-directed mutagenesis (Benoit
et al., 2006; van den Ent and Lowe, 2006).