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Gaylord et al. 10.1073/pnas.0407578101. |
Fig. 7. Wild type (WT), R406W mutant (RW), and noncomplementary N-terminal (N); double-stranded DNA sequences (~250 bases) generated from PCRs using WT and R406W DNA plasmids. All lanes are imaged on the same 1.2% agarose gel with a 1-kb DNA ladder for reference. Note an intervening lane was omitted between the ladder and DNA samples.
Fig. 8. Oligomer target sequences (D0, D1, D2, and D3) annealed with either a peptide nucleic acid (PNA) or DNA probe in 1 mM sodium acetate pH 6.5 and digested with S1 nuclease (0.75 units/m L, 37°C, 60 min, [PNA-FL/ssDNA] = 2 × 10-6 M) in the presence of S1 buffer (FL, fluorescein; ssDNA, single-stranded DNA). Samples were quenched with 10 mM EDTA and heated to 90°C for 10 min in the presence of urea loading buffer before loading on a preheated, 20% denaturing polyacrylamide gel run at constant power (30 W). Both 14- and 28-base single-stranded oligonucleotides were run as standards (S), and the gel was visualized by SybrGold staining (l ex = 365 nm).
Fig. 9. Noncomplementary (D3) and two-base mismatch (D2) oligomer target sequences annealed with the peptide nucleic acid-fluorescein (PNA-FL) probe and digested with S1 nuclease (0.5 units/m l, 37°C, [PNA-FL/ssDNA] = 2 × 10-6 M) in S1 buffer and quenched with 10 mM EDTA. Reaction samples were taken 20 and 40 min after S1 addition and heated to 90°C for 10 min in the presence of urea loading buffer before loading on a prewarmed, 20% denaturing polyacrylamide gel run at constant power (30 W). Both 14-and 28-base single-stranded oligonucleotides were run as standards (S), and the gel was visualized by SybrGold staining (l ex = 365 nm).
Fig. 10. Fluorescence spectra (selected from Fig. 1) of peptide nucleic acid-fluorescein (PNA-FL)/single-stranded DNA (ssDNA) upon addition and excitation (l ex = 380 nm) of the conjugated polymer (CP) ([CP] = 4 × 10-7 M and [PNA/ssDNA] = 1 × 10-8 M) in 30 mM potassium phosphate buffer (pH 7.4) at room temperature. The black curve corresponds to the complementary (wild type) DNA and the red curve to the noncomplementary (N-term) DNA targets.
Fig. 11. Fluorescence spectra (selected from Fig. 3) of oligomer targets (D0, D1, and D3, black, red, and green, respectively) annealed to peptide nucleic acid-fluorescein (PNA-FL). Spectra were obtained (l ex = 380 nm) upon addition and excitation (l ex = 380 nm) of the conjugated polymer (CP) ([CP] = 2.5 × 10-7 M and [PNA/ssDNA] = 1 × 10-8 M) in 30 mM potassium phosphate buffer, pH 7.4.
Fig. 12. Fluorescence spectra (from Fig. 5) of S1-treated oligomer targets (D0, D1, D2, and D3, black, red, blue and green, respectively) annealed to peptide nucleic acid-fluorescein (PNA-FL). Spectra were obtained (l ex = 380 nm) by adding conjugated polymer (CP) directly to the quenched reaction mixtures diluted ([CP] = 1.5 × 10-7 M and [PNA/single-stranded DNA] = 1 × 10-8 M) in 30 mM potassium phosphate buffer, pH 7.4.
Fig. 13. Fluorescence spectra (from Fig. 6) of S1-treated 249-base targets (WT, black, and R406W, red) annealed to peptide nucleic acid-fluorescein (PNA-FL). Spectra were obtained (l ex = 380 nm) by adding conjugated polymer (CP) directly to the quenched reaction mixtures diluted ([CP] = 1.4 × 10-7 M and [PNA/single-stranded DNA] = 1 × 10-8 M) in 30 mM potassium phosphate buffer, pH 7.4.