Caister Academic Press

Quantitative Real-Time PCR

Authors: N. A. Saunders
Abstract: Unlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the same in all samples and in the external quantified standards. Consequently, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples. Careful optimisation of the PCR conditions is therefore required. The use of probes in quantitative real- time PCR improves its performance and a range of suitable systems is now available. Generally quantitative real-time assays have excellent performance characteristics including a wide dynamic range, high sensitivity and accuracy. This has led to their use in a wide range of applications and two examples are presented. Viral quantification is now an important factor in the control of infection. The problems associated with virus quantification in cytomegalovirus (HCMV) infection are similar to those presented by other viruses. Quantitative PCR is finding an increasing role in the diagnosis of cancer. The assessment of c-erbB2/Her2/neu gene duplication is useful in predicting the disease prognosis in breast cancer. Several different real-time quantitative PCR protocols are available for these applications and have been applied successfully to their respective diseases.
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