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Quantitative polymerase chain reaction (qPCR) methods were first described in the 1980s (1), shortly after the discovery of PCR by Kary Mullis in 1983 (2). These earliest quantitative PCR methods relied on end-point analysis of the PCR product by staining, using a dye such as ethidium bromide, and visualising by gel electrophoresis. The intensity of the amplified band would be compared to standards of a known concentration to give a semi-quantitative result. Although the value of a quantitative result was recognised, this technique was time-consuming, lacked sensitivity and was not reliably quantitative. It wasn’t until some years later, in the 1990s, that real-time qPCR methods began to be described (1). Since this time, scientists have strived to find quicker, more effective ways to gain accurate quantitative data. Here we capture some of the more recent developments in probe-based technologies.
In real-time qPCR, the target DNA sequence is amplified and simultaneously quantified throughout the amplification reaction, during each PCR cycle. When the amplification reaction is in the log (linear) phase, the quantity of the PCR product is directly proportional to the amount of input nucleic acid target DNA sequence. Accumulation of the target DNA sequence in real-time qPCR (from now simply referred to as qPCR) is detected and measured using a fluorescent reporter molecule. As the quantity of target amplicon increases, so does the intensity of fluorescence emitted from the fluorescent label from either an intercalating dye or from a probe that is specific to a sequence within the amplicon. Reactions that contain a higher concentration of starting template take fewer cycles to accumulate a threshold concentration of PCR product, while those containing less starting template require more cycles; the threshold being the point at which the fluorescence resulting from amplified product is detectable above background fluorescence. The number of PCR cycles that elapse before the threshold is reached (Cq) is a measure of the input nucleic acid (see Figure 1). By comparing the results of samples of unknown concentration with a series of standards, the amount of template DNA in an ‘unknown’ reaction can be accurately determined. This approach is referred to as absolute quantification.
The quantity of target nucleic acid can also |
