DNA quantification using EvaGreen and a real-time PCR instrument

Abstract

DNA quantiWcation is an important, frequently used technique, and inaccuracies can result in failures with ligation, restriction, polymerase chain reaction (PCR),1 ampliWed fragment length polymorphism (AFLP), Southern blotting, and other techniques. DNA is most commonly quantiWed using absorbance at 260 nm, but because of the existence of many impurities, this can be an imprecise measurement and DNA levels can be more than 10 times overestimated in some cases [1]. QuantiWcation by agarose gel electrophoresis with a known amount of standard DNA [1,2] can provide more accurate data, but the procedures are complicated, the data often still are not accurate enough, and the technique is impractical for routine or high-throughput DNA quantiWcation [3]. Fluorescence spectroscopy using various DNA intercalating dyes is the most widely accepted technique for accurate DNA quantiWcation [4]. However, if the analysis is to be carried out with a Xuorescence spectrophotometer, a relatively large assay volume (e.g., 2 ml) is required [5], and this is impractical for small DNA samples and expensive dyes. Fluorescence can also be measured with a smaller volume of DNA sample using other instruments such as Xuorescent microplate readers [6], microplate Xuorometers [7,8], and transilluminator–microplate–CCD camera systems [9], but the instruments might not be readily available in most molecular biology laboratories. Real-time PCR instruments are common in molecular biology laboratories and can be used to measure Xuorescence. DNA intercalated with a dye emits Xuorescence at 520 nm under excitation at 490 nm, and with real-time PCR instrumentation the assay is carried out in PCR tubes, equivalent to spectrophotometer cuvettes used with a Xuo-