Evaluation of gold nanoparticles as the additive in real-time polymerase chain reaction with SYBR Green I dye

Abstract

Gold nanoparticles (AuNPs) have been proven to be able to improve the specificity or increase the efficiency of a polymerase chain reaction (PCR) when a suitable amount of AuNPs was used. However, there is still a lack of systematic evaluation of AuNPs in real-time PCR. In this study, DNA degradation and the fluorescence quenching effect of AuNPs were first tested in real-time PCR. Then two different kinds of Taq DNA polymerase, native and recombinant Taq polymerase, were employed to evaluate the AuNPs' effect on the threshold cycle (C(T)) values, standard curves and melting curves in real-time PCR. Different ratios of the amount of native Taq DNA polymerase to the amount of AuNPs were also tested. It was found that AuNPs could be applied in real-time PCR with correlation coefficient R(2)>0.989. The combination of 2.09 nM AuNPs with 3.75 U of native Taq DNA polymerase could make the amplification curves shift to the left and enhance the efficiency of the real-time PCR (0.628 39 without AuNPs compared with 0.717 89 with 2.09 nM AuNPs), thus enabling faster detection in comparison with those of control samples. However, no improvement ability of AuNPs was found in real-time PCR based on recombinant rTaq DNA polymerase. Besides, the results suggest that a complex interaction exists between AuNPs and native Taq DNA polymerase.

Real time PCR based on Fluorescent Quenching of Mercaptoacetic Acid-Modified CdTe Quantum Dots for Ultrasensitive Specific Detection of Nucleic Acids

“…However, the concrete mechanism is not still clarified well. Li, et al also report that gold nanoparticles [22][23] can improve the efficiency or specificity of PCR and shorten reaction times, the excellent heat-transfer property of nanoparticles may be responsible for improving PCR efficiency [23][24]. However, so far few report is closely associated with the effects of quantum dots on real-time PCR.…”

Effects of Superparamagnetic Nanoparticle Clusters on the Polymerase Chain Reaction

“…However, there have been a number of contradictory reports stating that gold nanoparticles have remarkably negative effects on PCR efficiency. Yang et al [8] reported that they could inhibit PCR efficiency at high levels of concentration, and suggested a complex interaction between gold nanoparticles and the Taq polymerase DNA synthetic enzyme. Vu et al [9] concluded that the surface interaction between gold nanoparticles and polymerase, rather than the DNA template or primers, inhibited PCR efficiency.…”

Enhancing the specificity and efficiency of polymerase chain reaction using polyethyleneimine-based derivatives and hybrid nanocomposites

“…Besides the conventionally used small molecular additives, such as formamide, 11 tetramethylammonium chloride 12 and its derivatives, 13 and betaine, 14 nanoparticles (NPs) have received considerable attention due to their unique physicochemical properties, which are significantly different from those of their bulk counterparts. Various NP systems, including nanogold, [15][16][17][18] carbon nanotubes (CNTs), 19 carbon nanopowder, 20 magnetic NPs, 21 semiconducting NPs, 22,23 and dendrimers 24 have been used as additives to improve the PCR specificity and efficiency. Nonspecific amplification problems can be overcome in the presence of NPs.…”