Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR
Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR
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DOI：
10.1093/nar/gng135
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发表时间：
2003-11-15
影响因子：
14.9
通讯作者：
Saint, CP
中科院分区：
文献类型：
--
作者：
Giglio, S;Monis, PT;Saint, CP
SYBR Green I （SG）作为插层染料广泛用于实时PCR应用，并包含在许多市售试剂盒中，其浓度未公开。SG与双链DNA的结合是非特异性的，需要额外的测试，如DNA熔化曲线分析，以确认特定扩增子的产生。熔融曲线分析的使用消除了琼脂糖凝胶电泳的必要性，因为特定扩增子的熔化温度（T-m）类似于电泳带的检测。当使用SG进行实时PCR多重反应时，只要T-m值有足够的差异，应该可以区分扩增子。使用市售试剂盒和内部SG母料对霍乱弧菌和肺炎军团菌进行实时多重检测，突出了性能特征的可变性，特别是在T-m分析中仅检测单一产品，而琼脂糖凝胶电泳中检测多种产品。检测到的T-m对应的扩增子G+C%较高且尺寸较大，提示在PCR过程中SG优先结合，导致在SG存在量有限的情况下，无法在多重反应中检测到多个扩增子。这对采用SG的实时PCR诊断分析的设计和常规应用具有启示意义。
SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations. Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon. The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T-m) of the specific amplicon is analogous to the detection of an electrophoretic band. When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T-m values are sufficiently different. Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T-m analysis but multiple products as assessed by agarose gel electrophoresis. The detected T-m corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting. This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.