Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

Figure 4

Visual IMSA assay and sensitivity evaluation of IMSA assay to test EV71. (a) Visual detection was performed with IMSA assay by adding HNB dye prior to amplification procedure. The color of sky blue demonstrates positive reactions while the color of violet demonstrates negative reactions. The number of the tube indicates IMSA reaction, respectively, as follows: 1: commercial Bst 2.0 DNA polymerase; 2: WT of Bst DNA pol LF; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant D540A; 7: LF mutant R412A; 8: LF mutant R412E; 9: LF mutant K416A; 10: LF mutant K416D; 11: LF mutant G310A-D540E; 12: LF mutant G310L-D540E; 13: negative control. (b) Fluorescence signals on real-time PCR instrument. Fluorescence values and curves were evaluated with Deaou-308C constant temperature fluorescence detection equipment. The reaction order in (b) table was arranged the same as tubes number in (a). The sign of “+” indicates positive reactions while “−” indicates negative reactions. Reactions 1–5 were able to amplify VP1 gene to detect EV71. The curves in different colors represent distinct proteins in IMSA reaction. Curve in black and “reaction 1” represent commercial Bst 2.0 DNA polymerase. Curve in green and “reaction 2” represent WT of Bst DNA pol LF. Curve in orange and “reaction 3” represent LF mutant D540E. Curve in pink and “reaction 4” represent LF mutant G310A. Curve in red and “reaction 5” represent LF mutant G310L.

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