Figure 6.

PCR amplification of UV-damaged DNA. (A) Human genomic DNA (K562) was damaged by exposure to UVC at a flux rate of 0.197 J/cm²/min. Two nanograms of damaged genomic DNA were amplified with primers specific for the Major and Precise Alu subfamilies (21). The PCR contained, inter alia, 2.5 U Taq Gold DNA Polymerase, 20 pmol each of the forward and reverse primers and, if applicable, 100 nM Dpo4. The forward primer was labeled at its 5′ end with the reactive fluorescent dye 6-FAM. Amplified fragments were separated by capillary electrophoresis and detected by laser-induced fluorescence of the incorporated dye-labeled primer. The electropherograms depict Alu element amplification products of the UVC damaged genomic substrate with Taq Gold DNA polymerase alone (1) or with a cocktail of Taq DNA polymerase and 100 nM Dpo4 (2). (B) Experimental details are the same as (A) except that the DNA was exposed to UVC at the same flux rate for 30 min and PCR was performed at 85°C, as noted in Materials and Methods. Results show the Alu element products obtained with AmpliTaq DNA polymerase alone (1) or with a cocktail of Taq DNA polymerase and 100 nM Ste (2) or Ain (3).