EliGene kits and Real-Time PCR instruments

EliGene® kits for real-time PCR analysis are divided according to detection chemistry to basic lines RT, UNI and LC virus, respectively.

In RT line, the TaqMan chemistry using hydrolyzing fluorescence probes is used in the combination with hot-start polymerase and ROX dye as a passive reference (see Figure 1A). The total reaction volume is 25ul when 5ul of isolated DNA is added directly to ready to use mastermix containing all necessary components for reaction. In RT kits, the fluorescence dyes FAM and HEX with excitation wavelengths of 495, 520 nm and emission wavelengths of 520, 550 nm together with non-fluorescence quencher BHQ1 are used. These kits are basically designed for ABI instruments (line 7000 and SteOne) and they are optimized on LightCycler480 instrument, however can be used with the other instruments that meet the spectral characteristics of used fluorescence dyes. The typical time of the run for RT kits is about 105 minutes.

In UNI line, the Molecular Beacons using hybridization fluorescence probes or TaqMan chemistry using hydrolyzing fluorescence probes are used in combination with hot-start polymerase (see Figure 1). The total reaction volume is 20 ul when 5 ul of isolated DNA is added directly to ready-to use mastermix containing all necessary components for reaction. The usage of uracil in the reaction mix enables UNG treatment to prevent the re-amplification of carryover-PCR products by removing any uracil incorporated into single- or double-stranded DNA. In UNI kits the fluorescence dyes FAM and HEX with excitation wavelengths of 495, 520 and emission wavelengths of 520, 550 together with non-fluorescence quencher BHQ1 are used. These kits are designed for almost all real-time instruments on the market. The typical time of run for UNI kits is about 60 minutes.

In LC virus line, the Molecular Beacons using hybridization fluorescence probes or TaqMan chemistry using hydrolyzing fluorescence probes are used in combination with one-step master mix for reverse transcription with consequent qPCR analysis (see Figure 1). The total reaction volume is 20/30 ul when 5/10 ul of isolated RNA is added to mastermix created by mixing of polymerase with reaction mix containing all necessary components for the reaction. The fluorescence dyes FAM, HEX and Cy5 with excitation wavelengths of 495, 520 and 649 and emission wavelengths of 520, 550 and 666 together with non-fluorescence quenchers BHQ1 and BHQ2 are used. The LC kits. These kits are designed for the majority of real-time instruments on the market. The typical time of run for LC kits is about 70 minutes.

Figure 1. Basic principle of TaqMan hydrolization probes (A) and Molecular Beacons hybridization probes (B) action.

LC and RT kits

The TaqMan probe hybridizes between the primers in annealing step and during elongation step probe is cleaved by exonuclease activity of Taq Polymerase. The Molecular Beacon probe forms beacon-like structure that opens to specific sequence during the hybridization in annealing step (B). “TaqMan” is registered trademark of Roche Molecular Systems Inc., “Molecular Beacon” is registered trademark of Public Health Research Institute Properties, Inc. (PHRI)