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Figure 2 | Malaria Journal

Figure 2

From: Deletion mutagenesis of large areas in Plasmodium falciparum genes: a comparative study

Figure 2

Overview of four currently used deletion mutagenesis methods. A) QuickChange™ site-directed mutagenesis method, B) ExSite™ site-directed mutagenesis method, C) Overlapping primer method, D) Inverse PCR method. The template plasmid (green and yellow, top panel) is denatured during the first step of the PCR reaction to allow the primers containing the desired mutation to anneal to the specific target sites. In the QCM method the primers completely overlap and the mutations are incorporated into both of the primers (A). The ExSite primers do not overlap at all; instead only one of the primers contains the mutation. One or both of the primers must also be phosphorylated (B). Partial overlapping at the 3' ends is characteristic of the overlapping primers. The mutation is present in both of the primers within the overlapping region (C). And finally, primers for the inverse PCR method do not overlap, but simply start at the opposite ends of the desired area to be deleted (D). The pink and blue arrows are the sense and antisense primers. Crosses on the primers represent the mutation sites and P is the single phosphorylated ExSite™ primer in B. The PCR conditions for the specific deletion of a ~400 bp parasite-specific insert from the 7.4 kb template (described here) are given in the table. The primers are extended at 68°C during which the desired mutations are incorporated and subsequently amplified. Two different linear PCR products are created during the PCR reactions, parental template and mutated DNA. The parental DNA is degraded by a Dpn I digestion step while the mutant DNA is circularized by blunt-end ligation. The newly formed mutant plasmids can subsequently be transformed into competent cells

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