Preliminary Study On The Enhancing Effect Of CdTe Quantum Dots For Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) technology is a basic experimental toolin the biomedical research. People have been working on the search of PCRenhancers since its invention. In this study, one specific kind of nanomaterials, CdTequantum dot, were employed toinvestigate potential enhancing effects on PCR.This thesis mainly analyzed from four aspects the possibility of the quantumdots as PCR enhancers. First, experiment models and conditions were set up toinvestigate potential effects of quantum dots as additive; Second, how quantum dotsaffected the specificity, sensitivity and stability of conventional PCR was studied;Third, how quantum dots affected the specificity, sensitivity and stability ofreal-time quantitative PCR (qPCR) was observed; Fourth, amplification fidelityunder quantum dots conditions was measured.(1) Set up of optimum conditions： The best working concentration of quantumdots was about1mg/ml for both conventional PCR and qPCR;(2) Effects of quantum dots on conventional PCR： CdTe quantum dotsincreased the yield, raised the sensitivity till100-fold, improved the specifity, andraised the stability (or repeatability) till the third round amplification.Besides,different quantum dots had different effects on PCR efficiency.Tree different haddifferent effects on PCR yields, but displayed similar effects on anneanlingtemperatures.(3) Effects of CdTe quantum dots on qPCR. First, under optimized conditi ons,CdTe quantum dots had similar effects for conventional PCR and qPCR, such asincreasing PCR yield,raising the sensitivity and specificity. Second, concentrationsof quantum dots forqPCR were tested. In most cases,0.4-1.0μl quantum dots (stocksolution1uM) generated similar best effects for both SYBR GreenI andEvaGreen-based qPCR system, but higher concentrations led to inhibition effects. Third,it was found that smaller sizes of quantum dots demonstrated better performance onPCR yield and sensitivity than those with larger sizes. Fourth, quantum dots alsoimproved the stability of qPCR.(4) Using rpsL system for fidelity assay, we found that quantum dots resμlted inlower fidelty, suggesting that quantum dots possess molecμlar toxicity. However,DNA sequencing of PCR products indicated that there were no apparent mutationsin the PCR products from quantum dots-faciliated reactions.The nolvelty of this study is： CdTe-based qPCR system was systematicallytested usingEva Green and SYBR GreenI fluorescent dyes and a new qPCR kitusingEva Greenwas developed.