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DNA Extraction and Quantitation for Forensic Analysts

Quantitative PCR

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Quantitative PCR (qPCR), sometimes referred to as real time PCR (rtPCR), is the most accurate, precise, and efficient method currently available for human DNA quantitation. It is an amplification process that detects and measures the accumulation (or reduction, depending on the kit) of fluorescent dyes as the reaction progresses.

The initial quantity of DNA in the sample is detected by monitoring the exponential growth phase of the reaction and measuring the cycle number at which the fluorescent intensity of the sample reaches a plateau. This cycle number is directly proportional to the quantity of DNA in the reaction. Analysis of the quantity of DNA in the sample is performed using software that compares the unknowns with the best fit regression line constructed from the standards.

Quantitative PCR is advantageous in that it can simultaneously assess the total amount of human DNA in the sample, but it also uses an internal positive control that can assess the level of inhibition that is present. Since the amount of internal positive control is consistent in each sample, if no signal is detected from this probe, inhibition is suspected to be present.

The most recently developed rtPCR kits go one step further and also estimate the quantity of male specific DNA in the reaction. If the ratio of human to male DNA is high (i.e. an abundance of female DNA is present) this can help the scientist better evaluate the sample for further testing prior to engaging in PCR.

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