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Fig. 5 | Genome Biology

Fig. 5

From: Zinc finger nuclease-based double-strand breaks attenuate malaria parasites and reveal rare microhomology-mediated end joining

Fig. 5

Copy number analysis by qPCR on genomic DNA. a Schematic overview of chromosomes 12 and 13. The centromere of chromosome 12 is shown in red. Binding sites for primer pairs used for qPCR are shown. Primer pair C1 amplifies the product over the cutting site of the ZFNs, while primer pairs L1 and R1 bind approximately 100 kb away from the telomeres on the left and right arm of chromosome 12, respectively. L2 and R2 bind around 8 kb away from the cutting site. N1 and N2 bind on the ‘control’ chromosome 13 and are used for normalization. b The ratio of the relative copy number of amplicons from both sides of the break point on chromosome 12 (L1 and L2 on left of break; R1 and R2 on right of break) is shown for parasites isolated from mosquito salivary glands (SG) compared with parasites isolated from midguts (MG). The copy number of the left side of chromosome 12 is strongly reduced in the SG sample for parasites expressing ZFNs in the midgut, whereas the right side is not affected. All individual values, including errors, are shown in Additional file 6. c The relative copy number of PCR products amplified over the break point shown for genomic DNA isolated from midgut oocysts (MG) and from salivary gland sporozoites (SG). Note the near absence of product in salivary glands from parasites where ZFNs are expressed before (SpZFN, TrapZFN) or during (Uis4ZFN) sporozoite entry into salivary glands. Positive and negative error is calculated from standard error of the mean from technical duplicates

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