DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

Abstract

DNA damage blocks DNA polymerase progression and increases miscoding. In this study we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n-1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n-1 deletions. In addition, we introduce the Mean Modified Efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens.

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

“…DNA polymerases have generally low bypass efficiencies at abasic sites (36 ), preventing amplification of DNA templates with abasic sites (37 ). However, when DNA polymerases read through abasic sites, sequence artifacts can be generated.…”

High-Throughput Sequencing-Based Mapping of Cytosine Modifications

“…In fCABSeq, 5fC is protected from bisulfite-mediated deamination by treatment with O-ethylhydroxylamine (EtONH 2 ) (Figure 3.1). While abasic sites are a problem for the pull-down approach described above, they are not a problem for base-resolution mapping as abasic sites read as thymine [93]. We also tested whether reduction by NaBH 4 (see redBS-Seq below) protected 5fC from bisulfitemediated deamination, but found that EtONH 2 achieves better protection for 5fC after bisulfite treatment.…”

Raman Spectroscopy: A Promising Technique for Analyzing Nucleic Acid Refractory to PCR Amplification

“…Despite the advantages, PCR presents drawbacks that perturb its reliability in aDNA analysis. One of the drawbacks arises from the inability of polymerases to bypass blocking lesions that involve misinsertions (for example, dAMP opposite abasic sites [30], deletions [31], and ring fragmentation of either the sugar or the base, resulting in inhibition of replication [32]. Another drawback of PCR is its excessive sensitivity to intra-laboratory contamination by exogenous modern DNA.…”

“…It principally centers on identifying naturally occurring and creating genetically engineered polymerases possessing low fidelity levels and adept at negotiating lesions. What lies at the root of the quest for more lenient polymerases is the (in)efficiency of Taq polymerase at bypassing blocking lesions [31,57]. But, that issue does not arise with the exceptionally accommodating Y-family polymerases that frequently exercise translesion synthesis (TLS) [58].…”