We tested several commercially available QPCR mixtures that either contained UNG or were supplemented with UNG from various sources. We observed that most of them provided an unreliable level of protection from carryover contamination when small fragments (240 bp (13); generally 1 kb (18)]. We observed, however, that the addition of stabilizers and a longer incubation time for the UNG treatment provided a much higher level of carryover protection ((Figure 1)). In particular, we noticed lot-to-lot variability that could be leveled out by modulating the length of the UNG incubation step. Increasing the UNG digestion time from 5 min (the manufacturer's recommended time) to 45 min led to satisfactory levels of carryover protection (at 37°C; (Figure 1)A). The UNG treatment retarded the amplification from 7 cycles (5 min incubation) to 14.9 cycles (45 min incubation). Thus, in this experiment, the longer UNG incubation step improved, at no further expense, the level of protection by two orders of magnitude: the remaining, amplifiable material was decreased from 1.3% to 0.012%±0.005% of the initial material. This remaining material, corresponding to dU-containing fragments, was not fully degraded, as quantified by gel analysis (see Supplementary Figure S1). Supplementation with additional UNG from various commercial sources did not provide an equivalent level of protection but increased the costs considerably (data not shown). We have used these conditions over a period of two years and observed significant lot-to-lot variations in the efficiency of carryover protection. We therefore regularly test the reagents directly in our UQPCR mixture with a titration curve of dT- or dU-containing fragments. In the experiment shown in (Figure 1)B, after a 15-min incubation, the amplification efficiency was 96%±2% and the ΔCp (Cp, the number of cycles required to reach a threshold level of fluorescence in the log phase of the amplification) between the dT and the dU-containing template was 12.6%±0.3% cycles, corresponding to the amplification of 0.021%±0.006% of the initial dU-template. Here carryover protection was relatively insensitive to variations in incubation times (see Supplementary Figure S1, panel B). Thus, in this reaction mixture, the activity and the amount of UNG were high enough to render a longer incubation step unnecessary, showing that the remaining amplifiable material did not result from incomplete UNG degradation. We further tested whether the degradation of the abasic sites could be a limiting factor by varying the length of the 95°C post-UNG incubation step. When this step was extended from 2 to 10 min, the amplification efficiency decreased from 96% to 90% (see Supplementary Figure S1, panel B). Even though the ΔCp between dT- and dU-containing fragments is larger, this does not correspond to a higher level of protection when the lowered amplification efficiency is taken into account. Thus, no additional protection was achieved by varying this parameter.Figure 1.
What could be the nature of the remaining material amplified from UNG-treated dU template when prolonged UNG treatment does not afford extra protection? If it contained remaining abasic sites, they should not stop DNA polymerization because Taq DNA polymerases are able to perform bypass synthesis across this lesion (19). Alternatively, amplifiable material could be reconstituted from the fragmented template molecules in the early steps of the PCR by jumping PCR. Both bypass polymerization and jumping PCR are a major source of base misincorporation with the potential to give rise to in vitro mutations (6,20). In ancient DNA analyses, modification of the DNA sequence of previously amplified PCR products would render the UQPCR approach worse than a lack of decontamination. Indeed, when the DNA sequence of the PCR product from a newly analyzed sample is identical to one that had been previously obtained in the laboratory, suspicions of carryover contamination are legitimate. However, if the novel sequences differ from those previously obtained, carryover contamination is unlikely unless the procedure for carryover protection is itself mutagenic. This prompted us to analyze whether the sequences of the molecules amplified from an UNG-treated dU-containing DNA fragment were identical to the starting sequence. Eleven clones of the PCR product were sequenced and not a single base modification was detected. This is not entirely surprising because the most common misincorporation that results from both bypass synthesis and jumping PCR should not cause sequence variation when abasic sites and strand breaks are produced at dU nucleotides. Regardless of the type of DNA polymerase used, the nucleotide that is most frequently incorporated in front of an abasic site is dA because it fits best in front of the gapped strand (20). Furthermore, when Taq DNA polymerases add a nontemplated nucleotide, this latter is also a dA (20). Thus, in most cases, a dA should be inserted in front of the position where a dU was formerly located, just as it would if the dU were intact.