PCR diagnostic assays are extremely sensitive and generally capable of detecting a small number of specific molecules. The standards used in such highly sensitive assays must contain an extremely low concentration of template, usually in the range of 0.1–100 copies per microliter, or 10−10–10−14 micrograms per microliter, assuming that the size of templates is in the range of 300–3000 base pairs. Concentrations within this range are much lower than the detection limit of all conventional methods for nucleic acid quantitation. Most researchers therefore quantitate the nucleic acid target in a much more concentrated solution (in the magnitude of 10−2–10−3 micrograms per microliter) using conventional methods such as UV spectrophotometry. This solution is then diluted to a PCR standard containing an appropriate number of targets. A major disadvantage of this approach is that the target can only be quantitated in the more concentrated form of the standard, but not in the final reagent. This approach, although commonly used in research laboratories, is not suitable for the manufacture and quality control of diagnostic products, which require more stringent accuracy, reproducibility, and stability.
CITATION STYLE
Wang, Z., & Spadoro, J. (1998). Determination of Target Copy Number of Quantitative Standards Used in PCR-Based Diagnostic Assays. In Gene Quantification (pp. 31–43). Birkhäuser Boston. https://doi.org/10.1007/978-1-4612-4164-5_3
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