Analysing Melting Curves with TaqMan probe

[Leowe]

Hey guys! Hi again! Just a little question +)

Is it possible to analysis melting curves with TaqMab probe? Or it possible only with refference dye like Syber Green or Eva green?

[danfive]

SYBR green only.

[Eva Green]

Originally Posted by danfive

SYBR green only.

I found this info in a previous Thread and I am wondering if it is a
still correct. Also because I was told recently to use Eva Green for Melting curve and HRM and I don't want to buy something useless

[danfive]

Didn't know about EVA green and the other saturation dyes, looks like its an improvement on SYBR Green for Hi-resolution melt curves. So you should be OK using it.

[danfive]

Some info from [Only registered users see links. ] :

EVAGreen

EvaGreen® dye is a green fluorescent nucleic acid dye with features that make the dye useful for several applications including qPCR, high-resolution DNA melt curve analysis (HRM)1, real-time monitoring of thermophilic helicase-dependent amplification (tHDA), routine solution DNA quantification and capillary gel electrophoresis. The DNA-bound dye has excitation and emission spectra very close to those of fluorescein (FAM) or SYBR® dye Green I, making the dye readily compatible with instruments equipped with the 488 nm argon laser or any visible light excitation with wavelength in the region. EvaGreen dye is extremely stable both thermally and hydrolytically, providing convenience during routine handling. The dye is essentially nonfluorescent by itself, but becomes highly fluorescent upon binding to dsDNA. EvaGreen dye is nonmutagenic and noncytotoxic by being completely impermeable to cell membranes, unlike SYBR Green I, which enters cell rapidly and is known to be a powerful mutation-enhancer (Ohta, et el. Mutat. Res. 492, 91(2001).


The unique properties of EvaGreen dye have made it particularly useful in quantitative real-time PCR (qPCR) application. Compared with the widely used SYBR Green I, EvaGreen dye is generally less inhibitory toward PCR and less likely to cause nonspecific amplification. As a result, EvaGreen dye can be used at a much higher dye concentration than SYBR Green I, resulting in more robust PCR signal.

Features:

Very Little PCR inhibition: Exhibit much less PCR inhibition than SYBR Green I via a smart "release-on-demand" DNA-binding technology.
Highly Sensitive: Low PCR inhibition of the dye permits a higher dye concentration to be used for much greater fluorescent signal and high-resolution melt curve analysis (HRM).
Nonmutagenic and noncytotoxic: Nonmutagenic and noncytotoxic by standard Ames test; completely impermeable to cell membranes (see below).

Compatible with Fast PCR protocol: Minimal interference to PCR makes it possible to significantly shorten the chain extension time.
Compatible with multiplex PCR: No dye migration from amplicon to amplicon when used at the recommended concentration
Unsurpassed Thermal Stability, Hydrolytical Stability and Photostability: No detectable dye decomposition in PCR buffer at 95-100°C for 48 hours; highly stable under either alkaline or acidic condition; withstand repeated freeze-thaw cycles
Spectrally similar to SYBR Green I: Compatible with all major brand qPCR instruments and enzyme systems

EVA Green flyer --> [Only registered users see links. ]

[danfive]

A lot of info for HRM from gene-quantification.com

---> [Only registered users see links. ]

Includes:

HRM Introduction
HRM Applications
HRM Instrumentation
HRM data normalization*shape & shift
HRM Workflow
HRM assay design manual
Mutation Scanning by High Resolution Melt Analysis: Evaluation of Rotor-Gene 6000 (Corbett Life Science), HR-1 and 384-well LightScanner (Idaho Technology)
High Resolution Melt - TALKs

HRM Dyes: LC Green SYTO9 Eva Green Quantace BEBO SYBR Green NEW !

Genotyping with PCR - How to choose the right approach (by Tevik Dorak in The Scientist)
High-Resolution Melting Curve Analysis Novel Strategies For PCR-Based Mutation Scanning and Genotyping (by Michael Hoffmann, Karine Raymond and Jochen Hurlebaus)

Assay: High-Resolution Melting & Unlabeled Probes (by GEN)
Mutation Scanning of the Cytidine Deaminase Gene by High-Resolution Melting Curve Analysis Using the LightCycler 480 System (by Roche Applied Science in Bionity.com)
HRM (by LTF Labortechnik)

HRM application papers

Further HRM publications by Roche Applied Science and Corbett Life Science