Digital LightCycler® 5x RNA Master
Tests performed using the Digital LightCycler® 5x RNA Master are based on the digital PCR technology used in the Digital LightCycler® System. The digital PCR technology enables detection and more sensitive and accurate quantification of nucleic acids. Performance of tests using the Digital LightCycler® 5x RNA Master relies on a generic reagent concept and is exclusive to the Digital LightCycler® System. Selective amplification of target nucleic acid from the sample is achieved by the use of target-specific forward and reverse primers provided by the customer. A thermostable DNA polymerase enzyme is used for both reverse transcription and PCR amplification. Digital LightCycler® 5x RNA Master is a PCR master mix which includes deoxyuridine triphosphate (dUTP), instead of deoxythimidine triphosphate (dTTP), that is incorporated into the newly synthesized DNA (amplicon).1-3 Any contaminating amplicon from previous PCR runs is eliminated by the AmpErase enzyme included in the master mix during the first thermal cycling step. However, the newly formed amplicon is not eliminated since the AmpErase enzyme is inactivated once exposed to temperatures above 55°C. The Digital LightCycler® 5x RNA Master and Digital LightCycler® System is capable of simultaneously detecting up to 6 fluorescent dyes which are described in the Digital LightCycler® User Assistance.
1. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990;93:125-8.
2. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995;373:487-93.
3. Mol CD, Arvai AS, Slupphaug G, et al. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995;80:869-78.
