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Applied and Environmental Microbiology, November 2008, p. 6563-6569, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00624-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase

Jeong Jin Choi, Jae-Geun Song, Ki Hoon Nam, Jong Il Lee, Heejin Bae, Gun A. Kim, Younguk Sun, and Suk-Tae Kwon^*

Department of Genetic Engineering, Sungkyunkwan University, 300 Cheoncheon-Dong, Jangan-Gu, Suwon 440-746, Republic of Korea

Received 14 March 2008/ Accepted 30 August 2008

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The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10^–6) than Taq DNA polymerase (11.98 x 10^–6). Uniquely, Neq DNA polymerase also amplified DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.

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DNA polymerase (EC 2.7.7.7) plays essential roles in cellular DNA replication and repair. In recent years, thermostable archaeal DNA polymerases have been identified in many species (6, 7, 11, 19, 20, 34, 35). On the basis of amino acid sequences, DNA polymerases can be classified into at least six distinct families (A, B, C, D, X, and Y) (27), and most archaeal DNA polymerases have been identified as members of family B, along with eukaryotic replicative DNA polymerases and Escherichia coli DNA polymerase II (6). Previously reported archaeal family B DNA polymerases from hyperthermophiles possess a 3'5' proofreading exonuclease activity and so are capable of carrying out PCR with higher fidelity than Thermus aquaticus (Taq) DNA polymerase. Another notable feature of archaeal family B DNA polymerases is the recognition of uracil in DNA templates, which causes DNA synthesis to stall (14, 22). The archaeal family B DNA polymerases have a read-ahead function concerning uracil detection, based on an N-terminal pocket specific for uracil (12). In addition, archaeal family B DNA polymerases interact with another deaminated base, hypoxanthine (13).

Archaea have been recognized as a third domain of living organisms, distinct from the Bacteria and Eukarya (38). From a phylogenetic perspective on the basis of rRNA sequences, Archaea have been classified into four phyla: Crenarchaeota, Euryarchaeota, Korarchaeota, and Nanoarchaeota (3, 17). Nanoarchaeum equitans, which belongs to Nanoarchaeota, is a nano-sized and hyperthermophilic anaerobe and is the first-known obligate archaeal symbiont, which grows on the surface of a specific crenarchaeal host, Ignicoccus sp. strain KIN4/I (17, 18). The 490,885-bp N. equitans genome is one of the smallest microbial genomes and has been completely sequenced (37).

N. equitans family B DNA polymerase (Neq DNA polymerase) is encoded by two open reading frames, which are separated by 83,295 bp on the chromosome, including a split mini-intein sequence (37). Recently, we reported on the temperature-dependent protein trans-splicing of the naturally occurring split mini-intein of Neq DNA polymerase and the properties of the protein trans-spliced Neq DNA polymerase (Neq C) and genetically protein splicing-processed Neq DNA polymerase (Neq P) (7). Both the Neq C and Neq P forms of Neq DNA polymerase showed the same properties, as expected.

Although Taq DNA polymerase is the most frequently used DNA polymerase in PCR, this enzyme has two general drawbacks: a target length limitation of the product that might be amplified and the low fidelity of the final product. To overcome these problems, DNA polymerase mixtures composed of nonproofreading and proofreading DNA polymerases have been generated; consequently, PCR amplification with improved yields and longer targets has been achieved (1, 2). Commercial DNA polymerase mixtures, or "long PCR" kits, typically consist of Taq DNA polymerase along with a lesser amount of an archaeal proofreading DNA polymerase. These mixtures also exhibit an enhanced fidelity compared to Taq DNA polymerase (9).

Here, we describe the optimum condition and substrate spectrum of PCR using Neq DNA polymerase. Interestingly, Neq DNA polymerase is the first archaeal family B DNA polymerase able to amplify DNA fragment in the presence of uracil (the deamination product of cytosine) and hypoxanthine (the deamination product of adenine). We also report that a mixture of Neq DNA polymerase and Taq DNA polymerase improves the performance of Neq DNA polymerase for long and accurate PCR. Finally, we demonstrate the practicality of a Neq/Taq DNA polymerase mixture in preventing carryover contamination in PCR.

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Enzymes and reagents.
Neq P, referred to here as Neq DNA polymerase, was prepared as described previously (7). The following commercial thermostable DNA polymerases were purchased: Pfu DNA polymerase (Stratagene), Vent DNA polymerase (New England Biolabs), KOD DNA polymerase (Novagen), and Taq DNA polymerase (Rexgene Biotech). Thermolabile uracil-DNA glycosylase (BMTU 3346 UDG) was purchased from Roche. Unmodified nucleotides (dATP, dCTP, dGTP, and dTTP) and deaminated nucleotides (dUTP and dITP [I is inosine; ribose-attaching hypoxanthine]) were obtained from Sigma-Aldrich.

DNA polymerase activity assay.
DNA-dependent DNA polymerase activity was assayed as described previously (7). The 50-µl reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 50 mM KCl, 0.01% bovine serum albumin (BSA), 200 µM each of dATP, dCTP, and dGTP, 20 µM dTTP, 0.5 µCi of [methyl-³H]TTP (30 Ci/mmol; GE Healthcare), 1.25 µg of activated calf thymus DNA, and enzyme solution. To determine whether a DNA polymerase could catalyze the incorporation of dUTP, the assay was also performed using [5-³H]dUTP (5 to 30 Ci/mmol; GE Healthcare) and dUTP instead of [methyl-³H]TTP and dTTP, respectively. After a 30-min incubation at 75°C, the reaction was terminated by placement on ice. An aliquot was evenly spotted onto a 23-mm-diameter DE81 filter paper disc (Whatman), and the dried disc was washed in 0.5 M sodium phosphate (pH 7.0) buffer for 15 min and in 70% (vol/vol) ethanol for 10 min. The disc was dried and transferred to a liquid scintillation vial containing 5 ml of toluene-based cocktail. Incorporated radioactivity was determined using a Beckman LS6500 scintillation counter. One unit of DNA polymerase is defined as the amount of polymerase that incorporates 10 pmol of [³H]TTP into an acid-insoluble product at 75°C in 30 min.

PCR efficiency assay.
Oligonucleotide primers that anneal to DNA (31) were synthesized for the PCR assays. The sequences of the primers are presented in Table 1. PCR buffer optimization experiments were performed with 1 U of Neq DNA polymerase in a 50-µl reaction mixture containing 5 pmol each of primers -1F and -1R, 200 µM deoxynucleoside triphosphates (dNTPs), and 30 ng of DNA as a template. The reaction buffer conditions are indicated in the corresponding figure legend. PCR was conducted as follows: 10 min at 95°C; 25 cycles of 1 min at 94°C, 1 min at 56°C, and 2 min at 72°C; and 10 min at 72°C.

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TABLE 1. Primers used for PCR assays

PCR experiments for extension efficiency in sizes of 1, 2, 4, 6, 8, and 10 kb were performed using 23 ng of DNA and 1 U of Neq DNA polymerase in the optimized PCR buffer. The cycling protocol was 3 min at 95°C; 25 cycles of 30 s at 94°C, 30 s at 56°C, and 12 min at 72°C; and 15 min at 72°C.

PCR assay in the presence of deaminated bases.
For comparison of the utilization of deaminated bases, PCR was carried out using five kinds of DNA polymerases (Neq, Taq, Pfu, Vent, and KOD DNA polymerases) in the presence of 250 µM dUTP (in place of dTTP) or 250 µM dITP/dGTP mixture (in a ratio of 1:9). The efficiencies of dITP incorporation by Neq and Taq DNA polymerases were determined through PCR in the presence of different concentrations of dITP (the dITP/dGTP mixture concentration remained at 250 µM). Each PCR procedure using commercial DNA polymerase was conducted under the buffer system and conditions provided by the manufacturer. The PCR procedure consisted of 1 min at 95°C; 25 cycles of 20 s at 94°C, 20 s at 56°C, and 1 min at 72°C; and 5 min at 72°C. -1 primers were used.

DNA polymerase mixture for long-range PCR.
Taq and Neq DNA polymerases were combined in various ratios (10:1 to 100:1) based on the unit of enzymes (2 U/µl:0.2 to 0.02 U/µl). Amplified fragments of 10 and 20 kb were obtained using -10 and -20 primer combinations, respectively. The PCR buffer (SuperTaq buffer II; Rexgene Biotech) for Taq DNA polymerase was used for mixtures of Taq and Neq DNA polymerases. After 3 min of the denaturation step at 95°C, PCR amplification was performed in 25 cycles of 1 min at 94°C, 1 min at 56°C, and 8 min (10-kb target) or 15 min (20-kb target) at 72°C.

Primer combinations for 1 to 8 kb were used for the comparison of extension efficiency of DNA polymerases in the presence of dUTP. The extension efficiency of Neq plus DNA polymerase (Taq/Neq DNA polymerase mixture; 10:1 ratio) was compared with those of Taq and Neq DNA polymerases. PCR was carried out for 3 min at 95°C; 25 cycles of 20 s at 94°C, 20 s at 56°C, and 8 min at 72°C; and 10 min at 72°C. Yields of amplification were compared using the area density tool of LabWorks 4.6 software (UVP).

Mixtures of Taq DNA polymerase with other archaeal family B DNA polymerases (Pfu, Vent, and KOD DNA polymerases) were also prepared in a ratio of 10:1 (2 U/µl:0.2 U/µl). For an apparent comparison, the -4 primer combination was exploited to produce a 4-kb fragment. PCR was performed at 3 min at 95°C; 25 cycles of 20 s at 94°C, 20 s at 56°C, and 4 min at 72°C; and 10 min at 72°C.

PCR fidelity assay.
pJR2-lacZ (5.7 kb), an expression plasmid containing the entire lacZ gene (34), was used as a template. An 835-bp fragment containing the 5' region of the lacZ gene was amplified using the following primers: Lac-B, 5'-NNNNGGATCCAATGATAGATCCCGTCGTTTTAC-3', and Lac-C, 5'-NNNNATCGATAATTTCACCGCCGAAAGGCGC-3' (BamHI and ClaI sites, respectively, are underlined). PCR was performed with Neq, Taq, Neq plus, and Pfu DNA polymerases in the optimized buffer or in the buffer supplied by the manufacturer for 1 min at 94°C; 25 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72°C; and 5 min at 72°C. The PCR products were digested with BamHI and ClaI, purified, and ligated with the 4.9-kb BamHI/ClaI fragment from pJR2-lacZ. Escherichia coli XL1-Blue was transformed with each ligate by electroporation and was then plated on LB agar plates containing 50 µg/ml ampicillin, 0.2 mM isopropyl-β-D-thiogalactopyranoside, and 20 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Pale blue and white colonies were counted as mutated plasmids, and blue colonies were considered to be intact plasmids.

Application to prevent carryover contamination in PCR.
A 4-kb DNA substrate and a 2-kb uracil-DNA substrate (contaminant mimic) were amplified using DNA as a template. PCR was carried out in a total volume of 50 µl containing 23 ng of DNA, 5 pmol each of the corresponding primers, Taq DNA polymerase, SuperTaq buffer II, and 250 µM dNTPs (dATP, dCTP, dGTP, and either dTTP or dUTP). PCR was performed at 3 min at 95°C; 30 cycles of 30 s at 94°C, 30 s at 56°C, and 3.5 min at 72°C; and 5 min at 72°C. The amplified products were purified using the QIAquick gel extraction kit (Qiagen).

To mimic carryover contamination, a small quantity (75 pg) of the 2-kb uracil-DNA substrate was mixed with the 4-kb normal DNA substrate (150 pg). The PCR mixture contained the mixed DNA substrate, 5 pmol of -2 primer combination for the uracil-DNA target, 5 pmol of -4 primer combination for the normal DNA target, 250 µM each of dATP, dCTP, dGTP, and dUTP, and 1 U of heat-labile UDG. Two kinds of DNA polymerases, Neq plus and Taq, were used in the reaction. After incubation at 25°C for 10 min, UDG was inactivated by heating at 95°C for 5 min, and then the following PCR cycles (20 to 30 cycles) were carried out: 20 s at 94°C, 20 s at 56°C, and 3.5 min at 72°C.

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Optimization of PCR amplification using Neq DNA polymerase.
The optimal buffer for PCR with Neq DNA polymerase was determined. To determine the optimum pH, PCR was performed in the pH range of 7.0 to 9.0 using Tris-HCl buffer. The DNA fragment could be amplified at pH 7.5 to 8.5 (Fig. 1a). In addition, PCR was carried out under various concentrations of MgCl₂ and KCl. The optimal MgCl₂ and KCl concentrations were 1.5 mM (Fig. 1b) and 50 mM (Fig. 1c), respectively. According to duplicate experiments, the optimal buffer for PCR with Neq DNA polymerase consisted of 20 mM Tris-HCl (pH 8.0), 1.5 mM MgCl₂, 50 mM KCl, and 0.01% BSA as a stabilizer.

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FIG. 1. PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb DNA fragment was performed in a 50-µl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl2, 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb DNA fragment was performed in a 50-µl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl2. (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb DNA fragment was performed in a 50-µl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.

Neq DNA polymerase could produce DNA fragments up to 10 kb (Fig. 2), indicative of an alternative reasonable PCR enzyme. Most archaeal family B DNA polymerases used in PCR are derived from euryarchaeotes, in particular from the genera Pyrococcus and Thermococcus (5). Only a few reports regarding PCR amplification have so far described the use of crenarchaeotal family B DNA polymerases (20, 23, 34). The present report is the first description of the successful application of a nanoarchaeotal family B DNA polymerase in PCR.

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FIG. 2. Extension efficiency of Neq DNA polymerase. The amplification of the DNA fragments was performed in a 50-µl reaction mixture containing the optimized PCR buffer for Neq DNA polymerase. Lane M, DNA molecular size markers; lanes 1 to 6, amplified DNA fragments of indicated target sizes (kb).

Utilization of dUTP and dITP by Neq DNA polymerase.
The dUMP incorporation activity of Neq DNA polymerase was compared with those of commercial DNA polymerases (Taq, Pfu, Vent, and KOD DNA polymerases). When [³H]dUTP was used instead of [³H]TTP, Neq and Taq DNA polymerases showed relatively little reduction in total incorporated radioactivity compared to Pfu, Vent, and KOD DNA polymerases (Fig. 3). The relative efficiency of dUTP utilization of Neq DNA polymerase was 74.9%, similar to that of Taq DNA polymerase (71.3%) and much higher than those of Pfu (9.4%), Vent (15.1%), and KOD (12.3%) DNA polymerases.

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FIG. 3. Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [3H]dUTP compared to [3H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.

PCR was conducted using the aforementioned DNA polymerases in the presence of deaminated bases. Neq and Taq DNA polymerases could catalyze PCR in the presence of dUTP or dITP (Fig. 4a). On the contrary, the other three archaeal DNA polymerases could not produce any amplified fragment, as expected from the results of the DNA polymerase activity assay (Fig. 3). dIMP incorporation by Neq and Taq DNA polymerases was further monitored via PCR with dITP/dGTP mixtures in different ratios (the concentration of the mixtures remained at 250 µM). Neq DNA polymerase was capable of amplification of the target fragment until the dITP concentration reached 50%, while Taq DNA polymerase could also amplify the fragment, even at a dITP concentration of 75% (Fig. 4b). The amplification yield of Neq DNA polymerase in the presence of dITP was lower than that of Taq DNA polymerase.

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FIG. 4. Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 µM dNTPs (lanes 1 to 5); 250 µM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 µM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 µM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.

Long-range PCR using Neq plus DNA polymerase.
The PCR amplification of a 10-kb DNA fragment was carried out using mixtures of Taq and Neq DNA polymerases in various ratios (10:1 to 100:1). The most efficient DNA amplification was observed with the ratio of 10:1 (Fig. 5a) and confirmed by three independent experiments. In addition, a 20-kb DNA fragment was successfully amplified by the 10:1 mixture (Fig. 5b). We named the 10:1 mixture of Taq and Neq DNA polymerases the Neq plus DNA polymerase.

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FIG. 5. Long-range PCR with mixtures of Taq and Neq DNA polymerases. DNA fragments of 10 kb (a) and 20 kb (b) were amplified using mixtures of Taq and Neq DNA polymerases at the indicated ratios. Lanes M1 and M3, DNA molecular size markers (1-kb ladder); lane M2, /HindIII DNA size markers; lane 1, Taq DNA polymerase; lanes 2 to 11, mixtures of Taq and Neq DNA polymerases in various ratios.

The extension efficiency of Neq plus DNA polymerase in the presence of dUTP was examined and compared with those of each Neq and Taq DNA polymerase. Neq plus DNA polymerase amplified DNA fragments up to 8 kb with a high yield (Fig. 6a), whereas Taq DNA polymerase was barely capable of amplifying the 6-kb uracil-DNA fragment and was incapable of the amplification of longer fragments. In the 4-kb amplified products, Neq plus DNA polymerase yielded 2.3-fold more than Neq DNA polymerase and 3.4-fold more than Taq DNA polymerase. To examine whether mixtures of Taq DNA polymerase with other archaeal family B DNA polymerases (Pfu, Vent, and KOD DNA polymerases) could produce a uracil-containing fragment efficiently, each mixture was prepared in a ratio of 10:1. The Vent/Taq DNA polymerase mixture barely amplified the uracil-DNA fragment, and the other two polymerase mixtures could not (Fig. 6b).

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FIG. 6. PCR amplifications with Neq plus DNA polymerase in the presence of dUTP. (a) Comparison of extension efficiency. The amplification of the DNA fragments was performed using Neq (lanes 1 to 5), Neq plus (lanes 6 to 10), and Taq (lanes 11 to 15) DNA polymerases. Target sizes are indicated on the gel. (b) Comparison of DNA polymerase mixtures. The amplification of the 4-kb DNA fragment was performed using Neq DNA polymerase (lane 1), Taq DNA polymerase (lane 2), Neq plus DNA polymerase (Neq/Taq) (lane 3), Pfu/Taq DNA polymerase mixture (lane 4), Vent/Taq DNA polymerase mixture (lane 5), and KOD/Taq DNA polymerase mixture (lane 6). Lane M, DNA molecular size markers.

PCR fidelity of Neq and Neq plus DNA polymerases.
The fidelities of the Neq and Neq plus DNA polymerases in PCR were measured and compared with those of Taq and Pfu DNA polymerases. Neq DNA polymerase exhibited a relatively high fidelity, with an error rate of 5.53 x 10^–6 (Table 2), which was calculated using the following equation: error rate = mutation frequency/(number of base pairs/duplication) (25). Neq plus DNA polymerase had an error rate of 7.97 x 10^–6 in the buffer for Taq DNA polymerase. Error rates of DNA polymerases increased in the following order: Pfu (2.67 x 10^–6) < Neq (5.53 x 10^–6) < Neq plus (7.97 x 10^–6) < Taq (11.98 x 10^–6). The fidelity of Neq DNA polymerase was approximately 2.2-fold better than that of Taq DNA polymerase and was worse than that of Pfu DNA polymerase. Neq plus DNA polymerase also showed better fidelity than Taq DNA polymerase.

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TABLE 2. Comparison of the fidelity of Neq, Neq plus, Pfu, and Taq DNA polymerases

Combination of Neq plus DNA polymerase and uracil-DNA glycosylase to control carryover contamination in PCR.
The application of Neq plus DNA polymerase to prevent carryover contamination was assessed. Newly amplified products containing dUMP are susceptible to UDG (21, 33), and PCR using these templates with dUTP and UDG is widely used as a contamination control technique (24, 32). As a result, all 2-kb uracil-DNA fragments were degraded by UDG, and the amplification yield of 4-kb DNA fragment by the combination of Neq plus DNA polymerase and UDG was higher than that by the combination of Taq DNA polymerase and UDG (Fig. 7).

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FIG. 7. Comparison of Neq plus and Taq DNA polymerases, in combination with UDG, in preventing carryover contamination in PCR. To mimic carryover contamination, the 2-kb uracil-DNAs (75 pg) were added to new PCR mixtures that contained 150 pg of the 4-kb target DNAs. The mixtures were preincubated at 25°C for 10 min in the presence (lanes 2 to 7) or absence (lane 1) of 1 U of BMTU 3346 UDG. After heating at 95°C for 5 min, the mixtures were used for normal PCR cycling in the presence of dUTP. PCRs were carried out using Taq DNA polymerase (lanes 2 to 4) and Neq plus DNA polymerase (lanes 1 and 5 to 7). Lane M, DNA molecular size markers; lanes 2 and 5, 20 cycles; lanes 3 and 6, 25 cycles; lanes 1, 4, and 7, 30 cycles.

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Up to now, the known archaeal family B DNA polymerases have been incapable of using dUTP as a substrate (22). The uracil-binding pocket present in archaeal family B DNA polymerases strongly interacts with uracil and hypoxanthine, potently inhibiting PCR in the presence of dUTP and dITP (13). In a previous paper, we reported the protein trans-splicing of the naturally occurring split mini-intein of Neq DNA polymerase and its properties (7). Interestingly, we recently found that Neq DNA polymerase utilizes deaminated bases and is also able to amplify DNA fragments using dUTP and dITP (Fig. 3 and 4). Neq DNA polymerase is, therefore, the first archaeal family B DNA polymerase that catalyzes PCR in the presence of dUTP and dITP.

To clarify why only Neq DNA polymerase, among archaeal family B DNA polymerases, does not exhibit the read-ahead function, the specialized pocket regions of archaeal family B DNA polymerases for uracil recognition were compared with the corresponding region of Neq DNA polymerase. We aligned the amino acid sequence of the N-terminal domain of Neq DNA polymerase with those of six archaeal family B DNA polymerases from the genera Thermococcus and Pyrococcus that contain the uracil-binding pocket (12). Residues involved in uracil recognition were highly conserved in the six aligned archaeal family B DNA polymerases, but these residues were not conserved in Neq DNA polymerase (Fig. 8). This result implies that Neq DNA polymerase cannot form the pocket structure capable of interacting with deaminated bases (12, 13).

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FIG. 8. Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo DNA polymerase (12). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

The rationale for the absence of a uracil recognition ability in Neq DNA polymerase is currently uncertain. So far, the DNA polymerase-based defense mechanism against uracil incorporation during DNA replication is known to be present exclusively in Archaea (36), despite the presence of DNA repair enzymes, including UDG (30) and dUTPase (15). The examination of the N. equitans genome has revealed a full set of DNA repair enzymes, including UDG and dUTPase, even though it is one of the smallest genomes (490,885 bp) (37). Therefore, we surmise that there might be no necessity for N. equitans to possess a supplementary uracil-binding pocket in its DNA polymerase to maintain the fidelity/integrity of the genome, since it has relatively enough repair enzymes compared to other archaeal organisms.

The peculiarity of Neq DNA polymerase lacking the uracil-sensing function might be useful in PCR. For example, Taq DNA polymerase, which is likewise deficient in that function, is commonly used in particular cases that require the incorporation of uracil and hypoxanthine, in spite of some inadequacies, such as low fidelity (2). Firstly, utilizing dITP in PCR appears to be an easy and effective method to prevent premature enzyme pausing in sequencing reactions and base compressions, which lead to abnormal migration patterns during polyacrylamide gel electrophoresis (10, 26). Secondly, incorporating dUMP into DNA is frequently used to control carryover contamination in PCR (24). In routine diagnostics, the presence or absence of a target sequence in clinical materials may be tested with PCR-mediated DNA amplification (28). However, the exquisite sensitivity of PCRs makes them vulnerable to carryover contamination, leading to false-positive results (8). Substituting dUTP for dTTP during PCR and subsequent UDG treatment seem to be one of the most effective procedures to address the problem (29). In those applications, Neq DNA polymerase might produce a more improved PCR product than Taq DNA polymerase, since Neq DNA polymerase has not only the incorporation activity of dUTP and dITP but also the proofreading activity.

On the other hand, the incorporation of dUMP into the amplicon by Taq and Neq DNA polymerases is not as efficient as dTMP incorporation, as shown previously (24) and presently (Fig. 3). Besides, Neq DNA polymerase has a drawback of yield (Fig. 4a) and Taq DNA polymerase has a target length limitation in utilizing dUTP (Fig. 6a). Under diagnostic conditions, target sequences to be amplified, such as the viral DNA genome, exist in trace amounts; PCR amplification with high yield is, therefore, necessary for sensitive detection. To overcome the limitations of those DNA polymerases, Taq and Neq DNA polymerases were combined in a ratio of 10:1 (Neq plus DNA polymerase) and then used in PCR. Neq plus DNA polymerase gave two- to threefold increased yield in the 4-kb amplification compared to each Neq and Taq DNA polymerase in the presence of dUTP (Fig. 6a). Moreover, the fidelity of Neq plus DNA polymerase was approximately 1.5-fold better than that of Taq DNA polymerase (Table 2). Finally, we applied Neq plus DNA polymerase to the PCR proposed to prevent carryover contamination with heat-labile BMTU 3346 UDG. In the prevention technique of carryover contamination, Neq plus DNA polymerase provided more enhanced efficiency of PCR amplification than Taq DNA polymerase (Fig. 7).

Even though Neq DNA polymerase belongs to the archaeal family B DNA polymerases, it exhibits a unique substrate spectrum. In spite of the relatively low fidelity compared to Pfu DNA polymerase, perhaps reflecting the absence of the uracil-binding pocket, the substrate spectrum of Neq DNA polymerase confers many advantages. In addition, Neq plus DNA polymerase surpasses Taq DNA polymerase, especially in the PCR application intended to deal with carryover contamination. Neq plus DNA polymerase might be useful in long-range DNA amplification (4, 16) and various PCR-based applications.

 
This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea.

 
* Corresponding author. Mailing address: Department of Genetic Engineering, Sungkyunkwan University, 300 Cheoncheon-Dong, Jangan-Gu, Suwon 440-746, Republic of Korea. Phone: 82 31 290 7863. Fax: 82 31 290 7870. E-mail: stkwon{at}yurim.skku.ac.kr

Published ahead of print on 12 September 2008.

J.J.C. and J.-G.S. contributed equally to this work.

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1
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Applied and Environmental Microbiology, November 2008, p. 6563-6569, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00624-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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