Eischeid Anne C
Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U,S, Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD, USA 20740.
BMC Res Notes. 2011 Jul 28;4:263. doi: 10.1186/1756-0500-4-263.
Real-time PCR can be carried out using either probes or DNA dyes. SYBR Green has been used the most, but it suffers from several drawbacks. Numerous other DNA dyes are commercially available, but with limited structural information. Dye behavior in real time PCR is difficult to predict, so empirical data are needed. In the work described here, a panel of 23 different DNA dyes--including green, orange, and red SYTO dyes, EvaGreen, and SYBR Green--were evaluated with respect to their performance in real time PCR.
Data were analyzed for reaction inhibition, effects on amplicon melting temperature, fluorescent signal strength, and reaction efficiency. This is the first report of reaction efficiency using alternatives to SYBR Green. Results indicated substantial variation in performance even within the SYTO dye family. EvaGreen and the SYTO dyes 13, 16, 80, and 82 performed better than SYBR Green in general, and high reaction efficiencies can be achieved using these dyes.
Empirical data were generated for 23 DNA dyes. This paper confirms and extends previous findings that among commercially available DNA dyes, EvaGreen and certain SYTO dyes are the most desirable alternatives to the commonly used SYBR Green in real-time PCR.
实时荧光定量PCR可以使用探针或DNA染料进行。SYBR Green是使用最为广泛的染料，但它存在一些缺点。市场上还有许多其他DNA染料可供选择，但关于它们的结构信息有限。实时荧光定量PCR中染料的行为难以预测，因此需要实证数据。在本文所述的工作中，对包括绿色、橙色和红色SYTO染料、EvaGreen和SYBR Green在内的23种不同的DNA染料在实时荧光定量PCR中的性能进行了评估。
分析了反应抑制、对扩增子解链温度的影响、荧光信号强度和反应效率的数据。这是第一篇关于使用SYBR Green替代品的反应效率的报告。结果表明，即使在SYTO染料家族中，性能也存在很大差异。总体而言，EvaGreen和SYTO染料13、16、80和82的表现优于SYBR Green，使用这些染料可以实现较高的反应效率。
生成了23种DNA染料的实证数据。本文证实并扩展了先前的研究结果，即在市售DNA染料中，EvaGreen和某些SYTO染料是实时荧光定量PCR中常用的SYBR Green最理想的替代品。
