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Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

Kuwahara M, Nagashima J, Hasegawa M, Tamura T, Kitagata R, Hanawa K, Hososhima S, Kasamatsu T, Ozaki H, Sawai H - Nucleic Acids Res. (2006)

Bottom Line: We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template.Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate.By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan. kuwahara@chem.gunma-u.ac.jp

ABSTRACT
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

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Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with (A) Taq DNA polymerase, (B) Tth DNA polymerase, (C) Vent(exo-) DNA polymerase, (D) KOD Dash DNA polymerase and (E) KOD(exo-) DNA polymerase. The x-axis indicates the kind of triphosphate analog used, and the y-axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.
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fig4: Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with (A) Taq DNA polymerase, (B) Tth DNA polymerase, (C) Vent(exo-) DNA polymerase, (D) KOD Dash DNA polymerase and (E) KOD(exo-) DNA polymerase. The x-axis indicates the kind of triphosphate analog used, and the y-axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.

Mentions: As shown in Figure 4, 20 combinations of enzyme and triphosphate (e.g. Taq/CPR and Vent(exo-)/CPN) were greatly affected by the template sequences. The PCRs using these combinations generated modified DNAs only from region I or II. Overall, it appeared that combinations resulting in lower relative yields likely depend on the template sequences. In contrast, of 130 combinations, the 26 combinations underlined in Figure 4 generated the products with higher relative yields (>70%) from both region I and II; PCR products were generated by only 2 (Tth/TAC and Tth/TPR) of the 52 possible combinations with family A polymerases but with 24 (Vent(exo-)/TAC, TAF, TPN, TPR, CAF, CA6, CG6, CCN; KOD Dash/TAF, TG6, TME, CAC, CAF, CA6, CG6, CME, CCN; KOD(exo-)/TAC, TAF, TPR, CAF, CG6, CME, CCN) of the 78 combinations with family B polymerases. These results suggest that the family B polymerases have broader substrate specificities than the family A polymerases. This is consistent with a previous report by Held et al. (28) describing the substrate properties of some C5-modified dUTPs with C≡C (triple bond) and C-C (single bond) linker arms for thermostable DNA polymerases. The present study clearly demonstrates a similar tendency in substrate specificities for the dCTP analogs.


Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

Kuwahara M, Nagashima J, Hasegawa M, Tamura T, Kitagata R, Hanawa K, Hososhima S, Kasamatsu T, Ozaki H, Sawai H - Nucleic Acids Res. (2006)

Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with (A) Taq DNA polymerase, (B) Tth DNA polymerase, (C) Vent(exo-) DNA polymerase, (D) KOD Dash DNA polymerase and (E) KOD(exo-) DNA polymerase. The x-axis indicates the kind of triphosphate analog used, and the y-axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636466&req=5

fig4: Relative yield of the modified DNAs generated by PCR using various triphosphate analogs together with (A) Taq DNA polymerase, (B) Tth DNA polymerase, (C) Vent(exo-) DNA polymerase, (D) KOD Dash DNA polymerase and (E) KOD(exo-) DNA polymerase. The x-axis indicates the kind of triphosphate analog used, and the y-axis indicates the relative yield of the PCR product. The black and white bars indicate the relative yield of the PCR product generated from amplifying regions I and II, respectively. The relative standard deviations were less than ±6% for all reactions.
Mentions: As shown in Figure 4, 20 combinations of enzyme and triphosphate (e.g. Taq/CPR and Vent(exo-)/CPN) were greatly affected by the template sequences. The PCRs using these combinations generated modified DNAs only from region I or II. Overall, it appeared that combinations resulting in lower relative yields likely depend on the template sequences. In contrast, of 130 combinations, the 26 combinations underlined in Figure 4 generated the products with higher relative yields (>70%) from both region I and II; PCR products were generated by only 2 (Tth/TAC and Tth/TPR) of the 52 possible combinations with family A polymerases but with 24 (Vent(exo-)/TAC, TAF, TPN, TPR, CAF, CA6, CG6, CCN; KOD Dash/TAF, TG6, TME, CAC, CAF, CA6, CG6, CME, CCN; KOD(exo-)/TAC, TAF, TPR, CAF, CG6, CME, CCN) of the 78 combinations with family B polymerases. These results suggest that the family B polymerases have broader substrate specificities than the family A polymerases. This is consistent with a previous report by Held et al. (28) describing the substrate properties of some C5-modified dUTPs with C≡C (triple bond) and C-C (single bond) linker arms for thermostable DNA polymerases. The present study clearly demonstrates a similar tendency in substrate specificities for the dCTP analogs.

Bottom Line: We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template.Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate.By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan. kuwahara@chem.gunma-u.ac.jp

ABSTRACT
We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Show MeSH