Quantification on the LightCycler

Abstract

Over the last 15 years, PCR has become an essential part of most laboratories involved in biomedical research. peR amplification turns a few attograms of a specific fragment of nucleic acid (far too little to be analyzed directly or used in biochemical reactions) into as much as a microgram of DNA. Everyone has heard the "quantitative peR is an oxymoron" joke, and it is not without some truth. By nature, an exponential amplification is not ideally suited to quantification. Small differences in amplification efficiency between samples can become huge differences in results when they are amplified through 40 doublings. Anyone working with quantitative peR who forgets this fact is in danger of making mistakes that are measured in orders of magnitude. Nonetheless, the years 1991-1998 saw a 10-fold increase in the number of papers using quantitative peR methods. Why then the continuing increase in the use of quantitative peR? It has a sensitivity five orders of magnitude better than the best blotting procedures and a dynamic range of 10 orders of magnitude. This unsurpassable sensitivity and range has made the work of turning peR into a quantitative tool worthwhile.