Figure 6

Telomeres in T. brucei cells prefer to have a 5′ end with a sequence of 5′ CCTAAC 3′. (a) A diagram showing the principle of the Telomere Oligo Ligation-mediated PCR (TOLP) assay. Seven different telomere guide oligos are separately ligated to intact genomic DNA. Only when the oligo is aligned immediately next to the 5′ end of the telomere can the oligo be ligated to the chromosome DNA. Subsequently the ligation products are amplified by PCR using a forward primer with the sequence of (TTAGGG)₃ and a backward primer with the sequence identical to the common regions of all guide oligos. The PCR products were then separated by agarose gel electrophoresis followed by Southern analysis using a telomeric probe. (b) TOLP was performed using genomic DNA isolated from WT BF cells that was treated with or without T7 exonuclease. The ethidium bromide-stained gel is shown on the top, and the Southern hybridization result using a telomeric probe is shown on the bottom. (c) Quantification of the TOLP signals from different guide oligos is shown. Average is calculated from three independent experiments. Error bars represent standard deviation.