Optimization Of Amplification Of GC-Rich Templates And Research On Gold Nanoparticles' Mechanisem

Nanoparticle PCR has gained extensive attention due to its ability to improve specificity in reamplification PCR systems. Later, gold nanoparticles (AuNPs) were demonstrated to enhance PCR's sensitivity and efficiency. However, amplification of GC-rich templates, as one of the bottle necks in PCR, has not been solved thoroughly. This article studied how to improve the amplification of some GC-rich fragments in human genome. After optimization of PCR programmes, amplification systems based upon Pfu DNA polymerase and Taq DNA polymerase were used to evaluate AuNPs'effect on the amplification of GC-rich templates. It was found that AuNPs could enhance the efficiency of these two amplification systems. When combined with some already known additives, AuNPs could also help to improve the amplification of relatively longer GC-rich template. However, AuNPs didn't show obvious advantages when compared with TAKARA's commercialized reagents. Besides, this article tried to understand the mechanism of AuNPs by studying the cause of smear bands in reamplification PCR systems. And these phenomena were thought to be caused by two reasons. First, complex secondary structures formed between products led to the artifacts in electrophoresis. Second, nonspecific amplification occurred due to some partially synthesized target fragments'mispriming. According to these results and the above hypothesis, the mechanism of AuNPs'improving specificity in reamplification PCR system was probably to inhibit the nonspecific priming events. It was also discovered that DNA polymerase had the strongest affinity for AuNPs in PCR systems through further experiments. Thus, probably AuNPs inhibit nonspecific amplification by interacting with DNA polymerase.