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. 2012 Mar;40(6):2793-806.
doi: 10.1093/nar/gkr1068. Epub 2011 Nov 24.

Highly Specific Unnatural Base Pair Systems as a Third Base Pair for PCR Amplification

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Free PMC article

Highly Specific Unnatural Base Pair Systems as a Third Base Pair for PCR Amplification

Rie Yamashige et al. Nucleic Acids Res. .
Free PMC article

Abstract

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.

Figures

Figure 1.
Figure 1.
Chemical structures of the unnatural Ds–Px pair and the natural A–T and G–C pairs. Functional groups (R), attached to the Px base via the propynyl linker, used in this study are summarized in the enclosed box.
Figure 2.
Figure 2.
PCR amplification involving the Ds–Px pairing with various DNA templates containing Ds for the determination of PCR conditions. (A) Scheme for PCR amplification experiments using 55-mer DNA fragments containing one Ds base, in the presence of dDsTP, NH2-hx-dPxTP and natural dNTPs. (B–D) Analyses by denaturing gel electrophoresis of PCR-amplified products after 15 cycles of PCR under different conditions by Deep Vent DNA pol (exo+) (B) and AccuPrime Pfx DNA pol (C,D), for the determination of the fold amplification at the end point.
Figure 3.
Figure 3.
Sequencing analysis of the PCR products after 15 cycles of PCR by each DNA polymerase: (A) Deep Vent DNA pol (exo+); (B) AccuPrime Pfx DNA pol; (C) Pfx50 DNA pol; (D) Pfu DNA pol; (E) Deep Vent DNA pol (exo); (F) TITANIUM Taq DNA pol. Sequencing reactions were performed in the presence of dPa′TP (left panels) or ddPa′TP (right panels) in each figure. The blue arrow indicates the original unnatural base position. The percentages indicated in the right panels for Ds-temp 1 and Ds-temp 4 are the retention rates of the Ds–Px pair in the amplified products after 15 cycles of PCR. The details of the calculation method for the retention rates are provided in the Supplementary Data section.
Figure 4.
Figure 4.
Real-time quantitative PCR amplification plots of each DNA template [(A) Ds-temp 1, (B) Ds-temp 4, (C) Cont-temp] and their linear standard curve analysis (D). PCR was performed with Deep Vent DNA pol (exo+) in the presence of 50 µM dDsTP and NH2-hx-dPxTP, 300 µM natural dNTPs, and different concentrations of DNA templates (20 fM, 200 fM, 2 pM, 20 pM).
Figure 5.
Figure 5.
Amplification efficiency and fidelity assessments of the Ds–Px pairing by 40-cycle and 100-cycle PCR amplifications (repeated 10-cycle PCR). (A) Scheme of the 40- or 100-cycle PCR amplifications of the Ds-containing DNA templates using dDsTP and modified-dPxTP, for the determination of the fold amplification, efficiency and selectivity of the Ds–Px pairing in PCR. (B) Scheme of the 40- or 100-cycle PCR amplification of the DNA template comprising only the natural bases using dDsTP and modified-dPxTP, for the determination of the misincorporation rates of the unnatural base substrates opposite the natural bases. The results are summarized in Tables 1 and 2. (C) Gel electrophoresis of the amplification products after each 10-cycle PCR (total 100 cycles of PCR) of Ds-temp 1 by the Deep Vent DNA pol (exo+) using 1 µM each primer, 30 µM dDsTP and NH2-hx-dPxTP and 300 µM natural dNTPs. A portion of each 10-PCR solution before (lanes T) and after each 10-cycle PCR (lanes +) was analyzed by 15% polyacrylamide denaturing gel electrophoresis, followed by SYBR Green II staining. M: marker (75-mer single-stranded DNA). (D and E) Sequencing analysis of the PCR products after 10, 40, 70 and 100 cycles of PCR by Deep Vent DNA pol (exo+) (D) and AccuPrime Pfx DNA pol (E). Sequencing reactions were performed in the presence of dPa′TP (left panels) or ddPa′TP (right panels). The blue arrow indicates the original unnatural base position. The percentage indicated in the right panels is the retention rate of the Ds–Px pair in the amplified products. The method for the calculation of the retention rates is provided in the Supplementary Data section. (F–H) Determination of misincorporation rates of the unnatural base substrates opposite the natural bases in Cont-temp after 10, 20, 40, 70 and 100 cycles of PCR by the Deep Vent DNA pol (exo+) using 30 µM dDsTP and NH2-hx-dPxTP and 300 µM natural dNTPs (first PCR). The second PCR products with the FAM-labeled 3′-primer and Cy5-hx-dPxTP were analyzed by 15% denaturing PAGE, and the DNA fragments were detected with FAM fluorescence (F) for the quantification of the total PCR products and with Cy5 fluorescence (G) for the quantification of the unnatural base misincorporated products. (H) Misincorporation rates calculated from the data are summarized in the panel.
Figure 5.
Figure 5.
Amplification efficiency and fidelity assessments of the Ds–Px pairing by 40-cycle and 100-cycle PCR amplifications (repeated 10-cycle PCR). (A) Scheme of the 40- or 100-cycle PCR amplifications of the Ds-containing DNA templates using dDsTP and modified-dPxTP, for the determination of the fold amplification, efficiency and selectivity of the Ds–Px pairing in PCR. (B) Scheme of the 40- or 100-cycle PCR amplification of the DNA template comprising only the natural bases using dDsTP and modified-dPxTP, for the determination of the misincorporation rates of the unnatural base substrates opposite the natural bases. The results are summarized in Tables 1 and 2. (C) Gel electrophoresis of the amplification products after each 10-cycle PCR (total 100 cycles of PCR) of Ds-temp 1 by the Deep Vent DNA pol (exo+) using 1 µM each primer, 30 µM dDsTP and NH2-hx-dPxTP and 300 µM natural dNTPs. A portion of each 10-PCR solution before (lanes T) and after each 10-cycle PCR (lanes +) was analyzed by 15% polyacrylamide denaturing gel electrophoresis, followed by SYBR Green II staining. M: marker (75-mer single-stranded DNA). (D and E) Sequencing analysis of the PCR products after 10, 40, 70 and 100 cycles of PCR by Deep Vent DNA pol (exo+) (D) and AccuPrime Pfx DNA pol (E). Sequencing reactions were performed in the presence of dPa′TP (left panels) or ddPa′TP (right panels). The blue arrow indicates the original unnatural base position. The percentage indicated in the right panels is the retention rate of the Ds–Px pair in the amplified products. The method for the calculation of the retention rates is provided in the Supplementary Data section. (F–H) Determination of misincorporation rates of the unnatural base substrates opposite the natural bases in Cont-temp after 10, 20, 40, 70 and 100 cycles of PCR by the Deep Vent DNA pol (exo+) using 30 µM dDsTP and NH2-hx-dPxTP and 300 µM natural dNTPs (first PCR). The second PCR products with the FAM-labeled 3′-primer and Cy5-hx-dPxTP were analyzed by 15% denaturing PAGE, and the DNA fragments were detected with FAM fluorescence (F) for the quantification of the total PCR products and with Cy5 fluorescence (G) for the quantification of the unnatural base misincorporated products. (H) Misincorporation rates calculated from the data are summarized in the panel.

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