Table 1.
The result of different concentrations enhancers for the amplification efficiency of moderate GC and GC-rich fragments by real-time PCR.
Fig 1.
PCR enhancers have different effects in amplification of fragment with moderate, high and super high GC-content.
Different PCR enhancers were tested at various conditions on the amplification efficiency by real-time PCR. (A-C) Representative amplification curve of the moderate GC fragment (53.8% GC), (D-F) high GC fragment (68.0% GC), and (G-I) super high GC fragment (78.4% GC).
Fig 2.
Enhancers such as betaine, sucrose and trehalose increase the thermostability of Taq DNA polymerase, but some enhancers function conversely.
Different PCR enhancers were used to test their effect on the thermostability of Taq DNA polymerase. Before subjected to thermocycling, PCR reaction mixtures were preheated at 95°C for 15 min (A) or 30 min (B). M: TaKaRa DL1000 DNA Marker. No preheating: PCR mixtures were not preheated. No enhancer: PCR mixtures without enhancers. All conditions were tested in duplicate. The original gel images are available in the S1 Raw images.
Fig 3.
Analysis of the resistance effect of Taq DNA polymerase to PCR inhibitor with PCR enhancers.
DNA fragments with moderate, high and super high GC-content were used for the analysis. (A) 0.0023 U heparin or (B) 0.0047 U heparin was added to the 20 μl PCR reaction with different PCR enhancers to amplify the moderate GC fragment. (C) 0.0094 U heparin or (D) 0.0375 U heparin was added to the 20 μl PCR reaction with different PCR enhancers to amplify the high GC fragment. (E) 0.0047 U heparin or (F) 0.0186U heparin was added to the 20 μl PCR reaction with different PCR enhancers to amplify the super high GC fragment. No heparin: PCR mixtures without addition of heparin. No enhancer: PCR mixtures without enhancers. All conditions were tested in duplicate. The original gel images are available in the S1 Raw images.
Fig 4.
Dose dependent effect of betaine in PCR amplification of long fragment with or without GC-rich region.
Various concentrations of betaine, sucrose and trehalose were tested for the amplification of a 10 kb (A) and a 15 kb λ phage DNA fragment (B). An 8 kb long DNA fragment containing a GC-rich region was subjected to amplification (C). All conditions were tested in duplicate. The original gel images are available in the S1 Raw images.
Fig 5.
Synergistic effect of betaine and sucrose in promote amplification of GC-rich region-containing long DNA fragment.
(A) 0.5 M betaine combined with different concentrations of trehalose and sucrose as PCR enhancers. (B) 1 M betaine plus different concentrations of trehalose and sucrose were used as PCR enhancers. All conditions were tested in duplicate. The original gel images are available in the S1 Raw images.