Real-time GUS analysis using Q-PCR instrumentation

Abstract

The development of new technology within biological sciences has resulted in a number of real-time PCR instruments that have become essential tools within molecular biology. This equipment has facilitated high throughput analysis of samples and optimal information gathering of completed PCR reactions for example estimating the copy number of a gene of interest that is inserted into particular genomes. Real-time PCR instruments frequently come with optional filter sets, e.g. the ALEXA filter set which has parameters in common with excitation and emission wavelengths of sodium methyl umbelliferone (NaMU) widely used in beta-glucuronidase reporter gene assays. Using these filter sets it has been possible to quantify and measure gus A activity of Ulmus procera SR4 in real-time removing the necessity for aliquots of reactions to be stopped by pipetting into carbonate buffer for each time point. The introduction of real-time GUS analysis leads to faster, more accurate and reproducible assays with reduced potential for pipetting errors, requires fewer manipulations and encourages high throughput analysis of inter-individual gene expression variation.