LIMITS AND PITFALLS OF SYBR GREEN DETECTION IN QUANTITATIVE PCR

Abstract

Both PCR and Real-Time qPCR are nowadays widely applicable techniques in diagnosis and research. InReal-Time qPCR, current detection methods are based on changes in fluorescence, which are proportional to the increaseof target. Fidelity of Real-Time RT-PCR is associated with its “true” specificity, sensitivity, reproducibility androbustness and a most important point in succeed a RT-qPCR is to choose the right detection chemistry. SYBR Green andTaqMan assays produce comparably dynamic range and sensitivity, while SYBR Green detection can be more precisethan the TaqMan probe detection if reaction specificity is perfected. When amplifying by the SYBR Green detectionsystem, we observed variations between genes in the specificity and reliability, proving that a unique amplificationsystem doesn’t exist, and that each experimental system has its own ideal conditions needing to be optimized. Therefore,we focused on the great dangers in evaluating SYBR Green quantitative results without complete optimization