Testing segmental gene conversion events.

<p>(A) Top, diagrams of Set_17 clone sequences drawn as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003502#ppat-1003502-g001" target="_blank">Figure 1</a>, with the binding locations of specific primers for donors 17-A (AF/AR) and 17-B (BF/BR) indicated. Below, PCR was performed using gDNA from pre-infection parasites (pre-infxn), and the first parasitaemic peak (08-08 and 09-08) and terminal samples (08-34 and 09-34) of primary clonal infections, using primer combinations AF-BR or BF-BR; the appearance of the AF-BR product only in the terminal gDNA reactions suggests that the junction A-B appeared over the course of infection and was present in the parasite population gDNA. Isolated plasmid clones were used as positive controls for each reaction. (B) Pre-infection gDNA was endonuclease digested using one of nine enzymes. Gel-separated, blotted DNA was hybridized to a probe corresponding to donor 17-A. Besides donor 17-A, this probe was expected to bind to two other <i>VSG</i>s, 17-B and 17-C. The expected positions of fragments corresponding to donors 17-B and 17-C are indicated on the figure with the letters ‘B’ and ‘C’ respectively; where the letter is in italics the fragment binds only part of the probe (hence resulting in multiple bands with a weaker signal). The remaining band(s) are highlighted with arrowheads; the strength of the signal indicates that these likely correspond with donor 17-A, the genomic locus of which is undetermined. (C) Set_14 PCR. Figure is laid out and annotated as panel A, with the binding location of primers specific for donors 14-A (AF/AR)and 14-B (BF/BR) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003502#ppat.1003502-Marcello1" target="_blank">[11]</a> indicated. PCR was performed using gDNA from pre-infection parasites (pre-infxn) and terminal samples of infections (01-32, 02-32, 03-32, 04-31, 05-31, 06-29), using primer combinations AF-AR, BF-BR and AF-BR. Isolated plasmid clones were used as positive and negative controls for each reaction. (D) Set_14 Southern Hybridization using a probe corresponding to donor 14-A. Besides 14-A, this probe was expected to bind to at least three other <i>VSG</i>s, 14-B, 14-C and 14-D. Figure is annotated as panel B, with the expected positions of fragments corresponding to donors 14-A, 14-B and 14-D indicated ‘A’, ‘B’ and ‘D’ respectively. In this case it is likely that the remaining band(s) correspond with donor 14-C, the genomic locus of which is undetermined.