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. 2015 Apr 16;43(13):6373–6383. doi: 10.1093/nar/gkv302

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Characterization of the elongation products produced. (A) Different forms of products of oligonucleotide elongation. Form 1. Tpg (large filled circle) linked to 1–3 radioactive dCMP (small filled circle). Form 2. Tpg linked to radioactive dCMP and non-radioactive dNMP (open filled circle) with the total length not exceeding 7 nt. Form 3. Tpg linked to13 nt-oligomer (Palindrome I) complexed with the template strand (shaded circles), the length of which is not to scale. Form 4. Tpg attached to the 13-nt oligonucleotide (which fold back to form a hairpin) without the template, representing the denaturation product of Form 3. Form 5. Tpg linked to 13-nt oligomer duplexed with the template strand of 14–16 nt, representing T7 endonuclease I digestion product of Form 3. Form 1′–5′ proteinase K digestion products of Form 1–5. Tpg was removed from the oligonucleotides. (B) The products of the first step of end patching based on TO65. The template was 65- (left and middle panels) or 89-nt (right panel) telomere DNA. The reaction conditions were as in Figure 2B except that the product was eluted from the G-25 spin column in deionized water. The eluted product was subjected to proteinase K (‘PK’) digestion, Exo III (‘EX’) digestion, or heat denaturation (‘Δ’). Left panel. Only radioactive dCTP (‘dC’) was included. Middle panel. Radioactive dCTP and non-radioactive dATP, dTTP and dGTP (‘all dN’) were included. The different forms of products identified in the SDS gels were marked. The sizes of marker proteins (in kD) are indicated to the right. (C) The products produced on templates of different lengths. Upper panel. The reactions were carried under the same conditions as described in (B) with all dNTPs added. The lengths of the template strands used are as indicated. In three cases, the incubation time was increased from 10 m to 30 and 60 m as indicated. Some products were further treated with T7 endonuclease I (‘Endo’). Lower panel. The products were heat-denatured before being subjected to electrophoresis. The various forms of the products are marked in the gels. The product, which was eluted for further analysis in (D) is marked by an asterisk. (D) Sizing the length of the oligonucleotide. Form 3 produced from the 89 nt template (marked with an asterisk in (C) was eluted from the gel slice in 0.1% SDS by soaking. The eluent was concentrated by ethanol precipitation, re-dissolved in 1 N NaOH, and incubated at 37°C for 2 h. After neutralization with HCl, the sample was electrophoresed in a 15% urea–PAGE at 20 V/cm together with three size markers—single-stranded DNA of TO13, TO16 and TO35 as indicated. The positions of the size markers were determined by UV absorption and EtBr staining. The position of the products was determined by autoradiography.