The Research Of Hot Start Gene Amplification Technique Based On Fluorescent Quantum Dots

Polymerase chain reaction (PCR) is an important nucleic amplificationtechnology in vitro which has been widely used in modern biology and medicalsciences. However, PCR technology has not reached its summit because it is stillfrequently impaired by low specific amplification and sensitivity arising from thenon-specific amplification products. Hot start(HS) PCR is one important techniqueto prevent those unwanted non-specific PCR products. The common approaches areinconvenient, prone to cross-contamination and not appropriate for high-throughputapplications, so it is necessary to explore a simple and effective HS strategy.Quantum dots (QDs) are quasi-zero-dimensional nanomaterials which aregetting more and more attention due to the excellent chemical and physicalcharacter. Recently, a hot start PCR amplification performance based on thewater-soluble CdTe QDs with Pfu DNA polymerase has been reported. However,whether QDs can trigger HS effects with other DNA polymerases is yet to beunderstood.In the present study, we studied the QD-triggered HS effects with fourhigh-fidelity and three conventional DNA polymerases and it was found that QDscould trigger a distinct HS PCR amplification performance with all the four testedhigh-fidelity DNA polymerases, and specific target DNA could be well amplifiedeven if the PCR mixture was pre-incubated for2h at50oC. On the contrary, the HSeffects were not prominent with all the three conventional Taq DNA polymerases.And then, the HS effects of QDs triggered were further researched by real-timefluorescence quantitative PCR (Q-PCR). It was found that QDs could significantlyimprove the specificity and sensitivity of Q-PCR using Pfu. A much specificamplicon was obtained even with low template concentration when the PCRmixture was pre-incubated for1h at50oC. This HS effect was comparable to thecommercial TaqHS. However, there were still no significant hot start effect with TaqDNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presenceof QDs, even after a1h pre-incubation at50oC before PCR. Finally, we made apreliminary study on the mechanism of QDs. The electrophoresis resultspreliminarily demonstrated that HS effect was not primarily due to the reduced polymerase concentration and QDs prefer to adsorb Pfu polymerases rather thanconventional ones, which might result in the QD-triggered HS effects on PCRperformance by using high-fidelity DNA polymerases.In summary, the QD-based HS PCR was very simple, universal and low cost,which could provide a better technical support for the subsequent studies.