Figure 2.

Principle of whole plasmid synthesis (WHOPS) and uracil-excision based site-directed mutagenesis (U-SM). (A) In WHOPS, two usually perfectly overlapping oligonucleotides are used to amplify an entire plasmid. WHOPS is used for site-directed mutagenesis by placing mutations (illustrated with an X) in the middle of the two primers. (B) Oligonucleotides for U-SM need only to overlap in the complementary region between the selected A- and T-nucleotides and only one primer needs to carry the mutation. (C) U-SM is compatible with making insertions (illustrated with a loop) and (D) deletions (missing sequence illustrated with a dashed line). (E) Larger insertions, such as in e.g. whole gene fusions, are made by a simple combination of the U-SM principle and uracil-excision based cloning - or even multiple fragments (not shown) as in the uracil-excision based PCR fusion principle.