HRM with SYBR Green So-called “new generation saturation dyes”, specifically LCGreen® and LCGreen Plus® (Idaho Technology), are promoted by some as essential for successful HRM analysis (Reed, Kent & Wittwer, 2007) although other dyes such as EvaGreen™ (Biotium Inc.) and SYTO®9 (Invitrogen) have been used with similar success (Krypuy et al 2006; Jeffery et al 2007; Wojdacz & Dobrovic; and others). Moreover, the use of SYBR® Green 1 (SYBR) for HRM is actively discouraged by some authors (Wittwer et al 2003; Reed, Kent & Wittwer, 2007). Liew et al (2004) state that SYBR can only be used for HRM after substantial modification of the protocol (including the use of GC-clamps, triple primers, and allele-specific PCR). By contrast, we and others (Price et al 2007; Pomprasert et al 2008) have found SYBR to be a very successful dye for HRM analysis that does not require any protocol modifications. We unhesitatingly recommend its use with a Rotor-Gene 6000. Prejudice against SYBR stems from early evidence collected on instrumentation that was not ideal for the task. Original assertions were based on experiments done with a pre-HRM era LightCycler™ capillary-based real-time analyzer (Roche Molecular Systems) using templates that included a low mass DNA size ladder (Wittwer et al 2003; Liew et al 2004). The reason SYBR was less successful than LCGreen was “not entirely clear” at the time, but a “dye redistribution” hypothesis was suggested (Wittwer et al 2003). According to this hypothesis, SYBR dye releases from low-temperature duplexes during melting and dynamically re-intercalates into neighboring duplexes that melt at higher temperature. This mechanism became the basis for the “dye saturation model” (Wittwer et al 2003, Liew et al 2004). According to this model, if sufficiently high concentrations of dye are used such that all binding positions on the DNA are occupied (i.e. saturated), then dye redistribution effects are minimized and greater melting curve resolution can be achieved. So-called “saturating dyes” were defined as those that can be used at concentrations sufficiently high to saturate all DNA binding sites without inhibiting the PCR. This model formed the basis for IP protection and a patent by Witter et al and Idaho Technology. In spite of the saturation model, mounting evidence clearly shows that it is not valid on the Rotor-Gene 6000 HRM instrument. This is true for a range of dyes, including SYBR (manuscript in preparation). In fact, SYBR used at standard non-saturating concentrations is highly suitable, as illustrated below for what is an especially challenging HRM application; the detection of a Class 4 SNP. We therefore urge users not to dismiss the use of SYBR for HRM. The reason saturating dye levels are not required for HRM on the Rotor-Gene 6000 (when apparently required on other instrument systems) is not clear. We have limited experience with competing instruments, however it must be noted that the centrifugal rotary format employed by the Rotor-Gene is distinctly different to other HRM instrumentation. Importantly, the Rotor-Gene has 25–50 times the thermal precision of other instruments and the shortest, most sensitive, and most uniform optical path. It also averages multiple readings for each data point reported at each discrete programmed thermal setpoint in a HRM. Surprisingly, Reed et al (2007) claim that the Rotor-Gene can only “approach high-resolution data quality by melting at slower rates”. Ironically, it may be that the slightly slower and more deliberate thermal stepping used by the Rotor-Gene is partly why it achieves superior HRM results without the need for a “saturating dye”. 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