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Figure 2
Measurement of G-overhang length by ligation-mediated primer extension. (A) Extension products obtained using T.thermophila DNA and various guide oligonucleotides: lane 1, input 21 nt guide oligonucleotide; lane 10, 30 nt marker showing the expected size of the input guide oligonucleotide + 9 nt extension product (a 14 nt overhang); lanes 2–9, purified rDNA minichromosomes; lanes 11–18, total macronuclear DNA; lanes 2 and 11, guide oligonucleotide TG1; lanes 3 and 12, TG2; lanes 4 and 13, TG3; lanes 5 and 14, TG4; lanes 6 and 15, TG5; lanes 7 and 16, TG6; lanes 8 and 17, mix of TG1–TG6; lanes 9 and 18, mix of TG1–TG6, no DNA ligase. (B) Ligation occurs specifically to the telomeric G-strand overhang: alkaline agarose gel showing restriction fragments labeled by ligation of 32P-labeled unique oligonucleotide/TG6 duplex to total macronuclear DNA (lanes 1 and 2) or purified rDNA telomeres (lanes 3–6): lane 1, ligation followed by BamHI digestion; lane 2, ligation followed by PstI digestion; lane 3, BamHI digestion prior to ligation; lane 4, Exo1 then BamHI digestion and ligation; lane 5, T4 DNA polymerase then BamHI digestion and ligation; lane 6, samples from lane 3 were digested with PstI to release the 1.2 kb telomeric fragment. (C) Organization of Tetrahymena rDNA: hatched boxes represent telomeres; shaded boxes labeled 3'nts and 5'nts represent the 5' and 3' non-transcribed spacer regions; open boxes represent the rDNA transcription units. B and P mark BamHI and PstI sites. (D) Measurement of overhang length using Euplotes crassus DNA: lane 1, primer extension products; lane 2, minus ligase control; lane 3, marker oligonucleotides. The 21 nt marker corresponds to the input guide oligonucleotide, the 30 and 58 nt markers correspond to the expected product if the guide oligonucleotide is extended only to the end of the 14 nt G-strand overhang or all the way through the duplex region of the telomere. The arrow marks excess guide oligonucleotide to which a single untemplated nucleotide was added by the T4 polymerase. A diagram showing the organization of Euplotes telomeres and the products expected from ligation-mediated primer extension is shown below.
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