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. 2006 Feb 18;34(4):1102–1111. doi: 10.1093/nar/gkj512

Figure 4.

Figure 4

Primer extension on CPD and abasic site-containing templates. Primer SSHTP2 (5′-GCG GTG TAG AGA CGA GTG CGG AG-3′) was annealed to the undamaged template, HTU50 (Un) (5′-CTC TCA CAA GCA GCC AGG CAT TCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′); the CPD-containing template, HMTT50 (CPD) (5′-CTC TCA CAA GCA GCC AGG CAT TCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′) (where the T-T CPD is underlined); or the abasic site-containing template, HTX50 (Abasic) (5′-CTC TCA CAA GCA GCC AGG CAT XCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′), (where X denotes the position of the abasic site). Primer extension assays utilized 10 nM of the three different primer/templates. The reactions containing the undamaged template (HTU50) was incubated at 60°C for 3 min, whereas the reactions containing the CPD (HMTT50) and abasic site (HTX50) templates were incubated at 60°C for 10 min. The concentrations of each enzyme utilized for the undamaged template (HTU50) reactions is as follows: Taq, 0.0025 U; Sso, 0.1 nM; Ste, 0.33 nM; Ssh, 0.4 nM; Ain, 0.2 nM; Ain/Sso, 0.5 nM; Ain/Ste, 0.5 nM. The concentrations of each enzyme utilized for the CPD-containing template (HMTT50) reactions was 50 times higher than that used for the undamaged template and is as follows: Taq, 0.125 U; Sso, 5 nM; Ste, 16.5 nM; Ssh, 20 nM; Ain, 10 nM; Ain/Sso, 25 nM; Ain/Ste, 25 nM. The concentrations of each enzyme utilized for the abasic site-containing template (HMTT50) reactions was five times higher than used for the undamaged template and is as follows: Taq, 0.0125 U; Sso, 0.5 nM; Ste, 1.65 nM; Ssh, 2 nM; Ain, 1 nM; Ain/Sso, 2.5 nM; Ain/Ste, 2.5 nM. Reactions were initiated by the addition of 100 µM of all four dNTPs (4) or 100 µM of individual dNTPs (G, A, T, C) (indicated below each lane). Replication products were separated on12%/8 M urea polyacrylamide gels and visualized by PhosphorImager analysis.