Quantification Of DNA Using Improved SYBR Green I RQ-PCR

Objective To investigate using improved SYBR green I real time quantitative polymerase chain reaction(RQ-PCR) technology to eliminate the contaminating fluorescence induced by the primer dimers(PDs), in ordei to establish more precise quantitaion of sample DNA content.Methods The target gene is B-actin. we could establish the improved SYBR green I real time quantitative PCR by measuring fluorescence at a temperature greater than the melting point of PDs but lower than desired gene after obtaining the melting temperature(Tm) of desired gene and PDs by analyzing the melting curve. Standard curve was constructed by using this way. Accuracy and stability of this improved technology were studied.Results Our research demonstrated that the contaminating effects of PDs fluorescence were largely eliminated with this improved protocol. A standard curve with a slope of -3.178866 and the correlation coefficient of-0.980535 was obtained by using our improved method. The linear range of PCR reaction was 5 logs (102-106). There was no significance (P>0.05) between measurement and expectation value of DNA copies in the examination of accuracy. The stability of the quantitation method were examined. The intra-run variability and inter-run variability were 1%, 3%, 2% and 2%, 4%, 4%.Conclusion Using this improved SYBR green I RQ-PCR strategy, one can eliminate the fluorescence induced by PDs. This offered a way of highly accurate and sufficiently sensitive and well stable quantification of DNA copies over a wide linear range. It is an cost-effective and feasible DNA quantitation method.