qPCR, HRM®, LAMP용 EvaRuby Dye | 고마바이오텍

특징

• EvaRuby는 자체적으로는 형광이 없으나 dsDNA에 끼어 들어가면 형광을 발광하는 red 형광 dye 입니다. 
• 480/613nm의 long stroke shift를 갖고 있어, EvaGreen과 동일한 green/blue 채널에서 excitation되고, 오렌지 채널에서 detection 됩니다. 
• real-time qPCR, melt curve analysis, LAMP, HRM (High Resolution Melting)에 사용됩니다.
• dye-based qPCR은 표적 특이성이 낮지만 melt curve analysis로 결과를 빠르고 쉽게 확인할 수 있는 장점이 있습니다. 반면에 probe-based qPCR은 높은 표적 특이성의 장점은 있지만 melt curve analysis와 호환되지 않습니다. EvaRuby를 probe-based qPCR과 결합하면 표적 특이성을 높이고 melt curve analaysis도 가능하게 됩니다. 
• 또한 probe 시그널과 독립적으로 발현되므로 PCR 증폭시 internal control로도 사용할 수 있습니다. (증폭이 잘 되었더라도 probe가 degradation되면 증폭된 서열에 잘 붙지 못하게 되는데, EvaRuby가 있다면 증폭중에 감지되고 melt curve로 식별될 수 있습니다.)

Figure 1. The effect of 1X EvaRuby™ Dye on the amplification and detection of the TFCR gene by a CAL Fluor® Red 610 hydrolysis probe in 10-fold dilutions of Human gDNA. Both EvaRuby™ and the CAL Fluor® Red 610 probe share the 610 nm emission channel. However, since EvaRuby™ is minimally excited by the Qiagen Rotor-Gene® Q Orange (585 nm) excitation channel used for the probe, the standard curve generated by the probe is practically unchanged by the presence of EvaRuby™. 

Figure 2. The effect of EvaRuby™ on Ct determination by hydrolysis probes. Three independent qPCR assays were performed for each probe dye using common probe-based qPCR settings. Each point is the Ct value determined by the indicated probe. Despite some overlap in the excitation or emission spectra, the Ct values obtained by the probes in a standard probe-based assay without EvaRuby™ (rings) are minimally different from the Ct values obtained when probes are used in combination with EvaRuby™ (circles). 

Figure 3. High Resolution Melt Data Difference Plot. EvaRuby™ Dye at 1X in Forget-Me-Not™ Universal Probe qPCR Master Mix (Cat. No. 31043) was used to detect the Applied Biosystems® MeltDoctor™ HRM Positive Control Kit. Data was acquired using the Qiagen® Rotor-Gene® Q, 0.1°C/2 sec, with a 470 nm excitation and 610 nm emission filter. Data analysis was performed with uAnalyzeSM (https://dna-utah.org) using baseline subtraction. EvaRuby™ Dye can clearly distinguish each homozygous variant as well as the heterozygous variant.