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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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Synthesis of C5-modified dUTP analogs: (i) H2N-(CH2)n-NH2 (n = 2, 4, 6), methanol, 50°C, 2 h overnight, followed by ethyl trifluoroacetate, triethylamine, room temperature (rt), 1.5–3 h; (ii) sodium methoxide, methanol, rt, 1.5 h; (iii) POCl3, N,N,N′,N′-tetramethyl-1, 8-naphthalendiamine, trimethyl phosphate, 0°C, 45 min, followed by n-tributylamine pyrophosphate, dimethylformamide, rt, 1 h; (iv) S-ethylthiourea, triethylamine, dimethylformamide, rt, 4 h.
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sch1: Synthesis of C5-modified dUTP analogs: (i) H2N-(CH2)n-NH2 (n = 2, 4, 6), methanol, 50°C, 2 h overnight, followed by ethyl trifluoroacetate, triethylamine, room temperature (rt), 1.5–3 h; (ii) sodium methoxide, methanol, rt, 1.5 h; (iii) POCl3, N,N,N′,N′-tetramethyl-1, 8-naphthalendiamine, trimethyl phosphate, 0°C, 45 min, followed by n-tributylamine pyrophosphate, dimethylformamide, rt, 1 h; (iv) S-ethylthiourea, triethylamine, dimethylformamide, rt, 4 h.

Mentions: The following commercial available thermostable DNA polymerases were purchased: Taq (Takara Bio), Tth (Toyobo), Vent(exo-) (New England Biolabs) and KOD Dash (Toyobo). KOD(exo-) DNA polymerase was generously donated by Toyobo. Natural 2′-deoxynucleoside-5′-triphosphates(dATP, dGTP, dCTP and TTP) were obtained from Roche Diagnostics. All modified nucleoside triphosphates used for PCR assays are listed in Table 1. TAL, TPR, CAL and CPR were purchased from TriLink BioTechnologies; TPA was from Geneact; and CPA was from JENA Bioscience. The other modified nucleoside triphosphates were synthesized (see Supplementary Schemes 1 and 2). Primers 1F, 1R, 2F and 2R were purchased from JBioS, and pUC18 template DNA was obtained from TaKaRa Biomedicals. Primer sequences and the sequences of the two amplifying regions on pUC18 used for PCR assays are shown in Figure 1. Primers P0, P1, P2, PA and templates TA, T0, T1, T2 and T3 used for kinetic experiments were purchased from Geneact. The sequences of these oligonucleotides are shown in Figure 2. Primers P1 and P2 and templates T1, T2 and T3 contained more than one modified dU [5-(2-(6-aminohexylamino)-2-oxoethyl-2′-deoxyuridine]. These oligodeoxynucleotides were obtained by chemical oligomerization using the C5-modified dU amidite prepared according to our published procedure (53). To detect and quantify extension products, the 5′ ends of 1F, 2F, P0, P1, P2 and PA were labeled with 6-carboxyfluorescein (6-FAM). Plasmid DNAs for sequence analyses were prepared using pT7Blue T-vector and DNA ligation kit ver. 1 from Takara Bio, a competent cell kit (Competent high DH5) from Toyobo and a plasmid DNA purification kit (LaboPass™ Mini) from Cosmo Genetech.


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)

Synthesis of C5-modified dUTP analogs: (i) H2N-(CH2)n-NH2 (n = 2, 4, 6), methanol, 50°C, 2 h overnight, followed by ethyl trifluoroacetate, triethylamine, room temperature (rt), 1.5–3 h; (ii) sodium methoxide, methanol, rt, 1.5 h; (iii) POCl3, N,N,N′,N′-tetramethyl-1, 8-naphthalendiamine, trimethyl phosphate, 0°C, 45 min, followed by n-tributylamine pyrophosphate, dimethylformamide, rt, 1 h; (iv) S-ethylthiourea, triethylamine, dimethylformamide, rt, 4 h.
© Copyright Policy
Related In: Results  -  Collection

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

sch1: Synthesis of C5-modified dUTP analogs: (i) H2N-(CH2)n-NH2 (n = 2, 4, 6), methanol, 50°C, 2 h overnight, followed by ethyl trifluoroacetate, triethylamine, room temperature (rt), 1.5–3 h; (ii) sodium methoxide, methanol, rt, 1.5 h; (iii) POCl3, N,N,N′,N′-tetramethyl-1, 8-naphthalendiamine, trimethyl phosphate, 0°C, 45 min, followed by n-tributylamine pyrophosphate, dimethylformamide, rt, 1 h; (iv) S-ethylthiourea, triethylamine, dimethylformamide, rt, 4 h.
Mentions: The following commercial available thermostable DNA polymerases were purchased: Taq (Takara Bio), Tth (Toyobo), Vent(exo-) (New England Biolabs) and KOD Dash (Toyobo). KOD(exo-) DNA polymerase was generously donated by Toyobo. Natural 2′-deoxynucleoside-5′-triphosphates(dATP, dGTP, dCTP and TTP) were obtained from Roche Diagnostics. All modified nucleoside triphosphates used for PCR assays are listed in Table 1. TAL, TPR, CAL and CPR were purchased from TriLink BioTechnologies; TPA was from Geneact; and CPA was from JENA Bioscience. The other modified nucleoside triphosphates were synthesized (see Supplementary Schemes 1 and 2). Primers 1F, 1R, 2F and 2R were purchased from JBioS, and pUC18 template DNA was obtained from TaKaRa Biomedicals. Primer sequences and the sequences of the two amplifying regions on pUC18 used for PCR assays are shown in Figure 1. Primers P0, P1, P2, PA and templates TA, T0, T1, T2 and T3 used for kinetic experiments were purchased from Geneact. The sequences of these oligonucleotides are shown in Figure 2. Primers P1 and P2 and templates T1, T2 and T3 contained more than one modified dU [5-(2-(6-aminohexylamino)-2-oxoethyl-2′-deoxyuridine]. These oligodeoxynucleotides were obtained by chemical oligomerization using the C5-modified dU amidite prepared according to our published procedure (53). To detect and quantify extension products, the 5′ ends of 1F, 2F, P0, P1, P2 and PA were labeled with 6-carboxyfluorescein (6-FAM). Plasmid DNAs for sequence analyses were prepared using pT7Blue T-vector and DNA ligation kit ver. 1 from Takara Bio, a competent cell kit (Competent high DH5) from Toyobo and a plasmid DNA purification kit (LaboPass™ Mini) from Cosmo Genetech.

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
Related in: MedlinePlus