Figure 3
Taq polymerase alone yields a S/N of 10 but can be improved Bridging PCR with Taq polymerase and the improvement of the S/N by addition of gp32 and (NH4)2SO4. The cut or intact templates was prepared by PCR with Taq polymerase and primers b1 and Jp on double-stranded plasmid pBS(-). BPCR was carried out with Taq polymerase and primers b1 and M13 pI. (See Figure 2 for location of primers and Table 1 for their sequence.) The other template was Phagescript phage particles. The 1 × PCR buffer for lanes 1–10 was 50 mM KCl, 10 mM Tris-HCl (pH 8.8 at 25°C), 1.5 mM MgCl2. The 1 × PCR buffer with (NH4)2SO4, (lanes 11 to 20) was 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25°C), 2.0 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Triton X-100 and 0.1 mg/ml bovine serum albumin. The ethidium bromide fluorescence image of the 1.0% agarose electrophoresis gel was photographed. The photograph was digitized and processed with Scion Image Adobe Photoshop software on a Macintosh computer. The image intensity was inverted.
Each lane has two rows which are labeled as cut, i.e. PvuII digested (upper row), or intact (lower row). The BPCR product bands are about 1261 bp, as expected from the template sequences (see figure 2). Lanes 1 to 5 show BPCR with Taq polymerase and no other additions. Lanes 6 to 10 show the enhancement of the S/N of BPCR by the addition of 0.01% gp32. Lanes 11 to 15, show the enhancement of BPCR by the addition of 10 mM (NH4)2SO4. Lanes 16 to 20, show BPCR with gp32 and (NH4)2SO4. The addition of both gp32 and (NH4)2SO4 did not improve the S/N. Each lane has two samples separately loaded into the two separate loading wells, one on each row. In the figure, the upper row is of source template cut with PvuII, and the lower DNA sample is of the same source template intact (without PvuII cut). The source template concentrations in the two samples were equal. Lanes 2, 3, 4 and 5 are serial template dilutions of lane 1. Lanes 7, 8, 9 and 10 are serial template dilutions of lane 6. Lanes 12, 13, 14 and 15 are serial template dilutions of lane 11. Lanes 17, 18, 19 and 20 are serial template dilutions of lane 16. Lane 21 contains DNA size markers made from Lambda DNA, BstE II digested, 125 ng. The source template concentrations of lanes 1, 6, 11 and 17 are adjusted to equal to 1 × 10-17 mol/μl PCR solution.