Here, we describe the isolation of novel chimeric polymerases with enhanced resistance to several environmental inhibitors with relevance for PCR applications. Starting from a diverse library of polymerase chimeras prepared by molecular breeding (33
) of the DNA pol I genes from members of the genus Thermus
, we performed CSR selections in the presence of complex inhibitor cocktails. We isolated a number of ‘specialist’ polymerases with specifically increased resistance to a given inhibitor such as Humic acid and clay-rich soil. From CSR selections using Neomylodon
bone powder as the inhibitor, a polymerase with broad spectrum of resistance emerged (). This polymerase (2D9) displayed a chimeric gene structure unlike any we had previously observed comprising segments from four different polymerases: Taq (1–109), Tos (110–388), Tth (389–456), Tbr (457–471) and Taq again (472–834), resulting in 81 mutations from the Taq consensus ( and Supplementary Data and Figure S4
). As with previously selected polymerases, the bulk of the main polymerase domain derives from Taq, presumably because a reaction buffer optimized for Taq was used for both selections and screening. Even subtle variations in reaction buffer composition can significantly affect PCR performance (39
). Although most of the other Thermus
enzymes (e.g. Tos, Tbr, Tfl and Tsc) showed good activity in Taq buffer, Tth and Tfi did not (data not shown). Thus a different set of polymerases may emerge using different buffer conditions (optimized e.g. for Tth) in the CSR selections.
All of the inhibitors examined in this study comprise complex, largely indescribable mixtures. Humic acid is a complex blend of poly-phenols (7
) while coprolites comprise a whole cocktail of substances that are potent inhibitors including bile salts, complex polysaccharides, bilirubin and hemin (4
). The tar samples comprise, in addition to inhibitors found in bone such as collagen and haem, diverse mixtures of polycyclic aromatic hydrocarbons (asphaltenes). Resistance to these diverse inhibitor cocktails may reflect resistance to a common inhibitory component present in all of these, but it is far from clear what this substance would be. Furthermore, 2D9 does display some selectivity in its resistance both in the degree of resistance (e.g. coprolites and Humic acids of different provenance) as well as in the absence of resistance (in comparison to Taq polymerase) to inhibition by complex samples containing related inhibitors such as human blood.
An alternative explanation would be that all of these substances share a common mechanism of inhibition. Indeed, one attractive explanation would be that the inhibitory activity of these different substance mixtures is due to their capacity for non-specific interaction with proteins and/or nucleic acids and the resulting sequestration of polymerase and template DNA from solution into an inaccessible form. Consistent with this hypothesis is the fact that the inhibition of both Humic acid and coprolite extracts can (at least partially) be overcome by the use of increased amounts of polymerase/primer oligonucleotides as well as the addition of non-specific protein (BSA) or DNA (salmon sperm DNA). Furthermore, this attenuating effect is additive (data not shown). Indeed, one of the problems faced during selections was a significant reduction of inhibitor potency in bacterial lysates. Aqueous CSR compartments during selection contain significant amounts of denatured protein, bacterial nucleic acids (mostly genomic DNA and rRNA) and membrane lipids from bacterial lysis and these were found to attenuate the inhibitory effect.
While it is unclear what the mechanism of non-specific adsorption would be, it is notable that Humic and Fulvic substances (as well as Asphaltenes) are known to form colloids. Indeed, most of the inhibitors appear as suspensions of fine particulate matter. Adsorption to the large surface area of colloid particles in suspension would provide an effective route to sequester significant quantities of protein and nucleic acids and offers a plausible mechanism of inhibition. Adsorbed to the surface of the colloids, the reagents would be inaccessible and unable to participate in PCR. Thus, 2D9 may simply display reduced interaction with colloids. However, inspection of surface charge or hydrophobicity distribution of Taq polymerase and a Phyre-threaded homology model (41
) of 2D9 do not reveal obvious localized differences, which could mediate such differential interactions, despite significant divergence at the sequence level.
Other potential explanations such as enhanced PCR performance of 2D9 (e.g. due to increased bypass of template abasic sites generated by thermocycling or exposure to chemicals in the inhibitor samples) or increased processivity do not appear to hold. Taq and 2D9 polymerase display a similar ability to bypass DNA lesions (Supplementay Data and Figure S6
), generate similar profiles of DNA products in primer extension reactions (Supplementary Data and Figure S7a
), and have similar processivity (both in the presence and absence of inhibitors), when measured under conditions that allow only a single cycle of DNA synthesis (Supplementary Data and Figure S7b
The discrepancy in inhibition profiles between primer extension reactions and PCR may (at least partially) be reconciled by consideration of the iterative nature of PCR, which may amplify fairly subtle differences, which would be challenging to detect in primer extension reactions. For example, an efficiency differential of as little as 10% in the presence of inhibitors could yield differences of up to 20-fold over a 30 cycle PCR. Inhibitor action during PCR thermocycling may also differ from extension reactions because of e.g. inhibitor effects on polymerase stability, primer/template rebinding or because of inhibitor action is temperature dependent.
The 2D9 polymerase showed greatly enhanced resistance (up to 47×) in the presence of some environmental inhibitors, notably clay-rich soil and tar-impregnated bone material, while showing enhanced resistance (5–15×) to the inhibitory effects of a broad range of other complex inhibitors such as coprolites, cave sediment, bone powder, peat and pure Humic acid (). At the same time, it retained an ability to function under more challenging PCR conditions, like amplifying ancient DNA extracted from cave–bear bone at limiting dilutions (). However, its sensitivity to amplify low-copy number targets (e.g. in ancient DNA PCR), although essentially identical to conventional Taq, was lower than that of an engineered hot-start variant of Taq (AmpliTaq Gold) (data not shown). Furthermore, when we examined AmpliTaq Gold (ATaqG) resistance to inhibitors we found that, although 2D9 outperforms ATaqG on most inhibitors (a), ATaqG is generally much less sensitive to inhibition than standard Taq, although based on the same enzyme. ATaqG is a chemically modified version of Taq DNA polymerase. The chemical modification renders the enzyme inactive at room temperature but high temperature and low pH (encountered in Tris-buffered PCR at 92–95°C) reverse the chemical modification and restore enzyme activity resulting both in hot-start PCR as well as a continued release of active enzyme during PCR thermocycling, which increases yields of specific APs. Amplification of low-copy number targets is especially vulnerable to side-reactions caused by extension of primers mis-annealed to non-target sequences or themselves (primer–dimers), which preferentially form at low temperatures. Hot-start PCR prevents extension of such illegitimate priming events by delaying polymerase activity until proper primer annealing temperatures are reached. There must therefore be also a distinct advantage in hot-start PCR in relation to inhibition sensitivity. Indeed, when we re-examined the resistance profile of 2D9 using a manual hot-start procedure, we found that hot-starting increased the already high resistance of 2D9 to inhibition by at least 2-fold (b).
Figure 6. Inhibition and the effect of hot-start. (a) Inhibition profiles of 2D9 versus AmpliTaq Gold 360 (ATaqG) in PCR. The effect of various inhibitors (SP61: bone dust, Tar, Soil, 1736; cave sediment) on polymerase activity of 2D9 and ATaqG as determined by (more ...)
The fact that a simple ‘manual’ hot-start further improved the already substantial resistance of 2D9 suggests that hot-starting contributes to ATaqG’s improved resistance compared to ‘standard’ Taq. This in turn indicates that the mechanism of inhibitor action may, in addition to the proposed sequestration of reagents, involve the promotion of mispriming events during early PCR cycles. Presumably, the proposed sequestration of template DNA and primers renders even abundant templates effectively low-copy number targets, which may then benefit from the increased specificity of hot-start amplification. Provided that there are no inhibitor absorbing components in the proprietary ATaqG buffer or that residual chemical modification of ATaqG does not alter its propensity to interact with the inhibitor cocktails, a further contribution to inhibitor resistance would likely derive from continued release of active enzyme during the course of PCR. Indeed, the resistance gain by (manual) hot-start (average: 2×) is significantly smaller than the differential between standard Taq and ATaqG (average: ~5×). The development of a chemical hot-start capability for 2D9 should allow disentanglement of these disparate effects and, by analogy with ATaqG, would be expected to lead to significant further increases in resistance.
In conclusion, CSR selection for resistance to complex environmental inhibitors yielded 2D9, a chimeric polymerase with significant resistance to inhibition by divergent organic and inorganic inhibitor cocktails such as coprolite, bone dust, soil, Humic acid and tar. The broad spectrum resistance of 2D9 to a variety of environmental inhibitors is promising benefits in a number of PCR applications where such inhibitors are present. However, but the full realization of the potential of 2D9 may require the development of a chemically- or antibody-based hot-start capability for this polymerase. The fact that just a single round of CSR selection yielded a polymerase with a substantially and broadly increased resistance underlines the power of the CSR method for the isolation of novel polymerase phenotypes.