2.3. PCR-Based Methods for Uracil Localization within DNA

To localize uracils within a target DNA sequence, several PCR-based methods are available that can provide either an indication for the presence of U:G pairs, or exact localization (even with single-base resolution), or accurate quantitation (within the target sequence) of the uracils. The first published technique to detect C:G to T(U):A transitions due to cytosine deamination in DNA was the differential DNA denaturation PCR (3D-PCR) [97]. This technique relies on the lower denaturation temperature (Td) of DNA templates with higher AT content. It applies gradiently lowered Td in the PCR reactions, amplifying a specific target sequence defined by the two PCR primers. The specific PCR product could be detected already at lower Td in those cases where some C:G to T:A transitions happened in the template DNA within the amplified region [97]. Later, this 3D-PCR technique was applied in combination with a UNG inhibitor, UGI, to detect uracil-DNA intermediates of APOBEC3A-catalyzed cytosine deamination in a reporter plasmid DNA [22].

Almost at the same time as the publication of 3D-PCR, the Gearhart group applied a combined in vitro reaction of UNG and APE1 to detect uracil in an exogenous plasmid from which AID was expressed in bacteria [98]. They could show AID induction-related increases in the number of nicks by a UNG/APE reaction on alkaline agarose gel, and could locate the uracil moieties on the non-transcribed strand using a denaturing Southern blot. Furthermore, they also applied polymerase β without the addition of dNTPs, just using its 5′dRP hydrolyzing function to introduce nicks to the site of the uracil. Then, they applied primer extension on the nicked template using a specific and biotinylated primer that results in dsDNA end that is appropriate for blunt end adapter ligation. By clonal sequencing of the products of this ligation mediated PCR (LM-PCR), they could localize the original positions of uracils within the non-transcribed strand of the AID-expressing plasmid with single base resolution [98]. Later, they further developed this technique and successfully detected uracils in the immunoglobulin genes of ung⁻deficient AID expressing B cells as compared to ung⁻deficient Aicda^−/− cells (ung⁻deficient chicken DT40 either overexpressing the chicken AID or Aicda^−/− clones, and B220+GL7+ spleen cells from ung^−/− and ung^−/−, Aicda^−/− mice that were either immunized right before the cells were isolated, or the isolated cells were stimulated ex vivo with LPS and Il-4) [99].

Another PCR-based approach simply quantifies the difference between the amounts of intact templates in the samples pre-treated with UNG alone or UNG + APE as compared to the non-treated one. Such quantification could be performed by qPCR, and also by the more convenient digital PCR techniques (such as digital droplet PCR (ddPCR)). In the ex-ddPCR method, samples are treated with UNG, and the fraction of amplicons containing at least one uracil on each strand is determined from positive PCR counts in the treated and un-treated samples [100]. It was shown that the viral gag gene accumulates uracils only in monocyte-derived macrophages (MDM), but not in T cells.

While these approaches above rely on specific enzymatic reactions by UNG and APE, a fully independent PCR-based method utilizes the altered sensitivity of archaeal DNA polymerase Pfu and its V93Q mutant version for the uracil-containing templates [27]. The structural basis and the functional consequences of binding of the archaeal family B polymerases to uracil bases in the template DNA strand has already been well described [101]. While a single uracil base can eventually stall DNA synthesis by wt Pfu polymerase, V93Q mutant Pfu preserves its activity even on fully uracil substituted templates [102]. Applying wt and V93Q mutant Pfu in parallel PCR reactions using the same template dilution series, from the difference between the corresponding Cq values, the uracil can be quantified within the given template region defined by the two PCR primers [27].