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Principles of PCR

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Molecular Diagnostics

Abstract

PCR is used to generate large amounts of nucleic acid from small amounts or target, using either conventional or real-time technology. Essential reagents are a thermostable DNA-dependant DNA polymerase, synthetic oligonucleotide primers, dNTPs and a balanced buffer system. The target can be isolated from various different source materials, and the PCR reaction is automated. The (c)DNA sample must reach a certain degree of purity or needs to be free of components that influence the polymerisation reaction. PCR is extremely sensitive and, when the parameters are correct, extremely specific. This allows a wide variety of applications in various clinical fields. A PCR consists of a number of repetitive cycles of alternating denaturation, annealing and extension. The hereby synthesised products (amplimers) can be characterised with a wide variety of techniques.

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Notes

  1. 1.

    A complete PCR on a full-length cDNA target of 30 kb can take 24 h; one cycle 30 min.

  2. 2.

    The four phases that we distinguish in the amplification curves should not be confused with the four phases often encountered in the popular literature that is depicted with a linear fluorescence axis. Although ground phase and plateau phase are defined similarly, the often encountered take-off phase and linear phase occur at the cycle numbers that describe the exponential phase and transition phase, respectively. Therefore, the so-called linear phase should not be confused with the exponential phase. It is in fact the transition phase and using these data for Cq read-out will lead to biased results.

  3. 3.

    Baseline is commonly used to describe the fluorescence coming from the fluorochrome used for monitoring the PCR but is not dependent on the ongoing amplification. The term background is used for the fluorescence and light from other sources like the plastics and reflection. The system software is correcting for the background light. The reported fluorescence can be either baseline corrected by the system software or not. In the latter case, the reported data are referred to as raw fluorescence data.

  4. 4.

    The TaqMan™ hydrolysis probe is designed to increase the specificity and sensitivity of real-time PCR. The method was developed in 1991 by Cetus Corporation and further refined by Applied Biosystems. TaqMan™ is a registered trademark, protected by patents. Instead of TaqMan™, we use the functional name, hydrolysis probe. The same holds for TaqMan® MGB, which is also protected by patent. Applied Biosystems can synthesise your MGB-probe. Self-designed hydrolysis probes can be ordered at many companies.

  5. 5.

    In many laboratories, the terms standard or calibration curve are not so strictly discriminated. In this book, we use ‘calibration curve’ to indicate that the Cq value of an unknown sample on the Y-axis is used to determine directly the target quantity on the X-axis (read-out using the straight line constructed by dilution series). The term ‘standard curve’ is used to indicate the method of efficiency determination by using the slope of the straight line constructed by dilution series. It is also used to calculate the target quantity by using efficiency and Cq outcomes.

  6. 6.

    In various sub-disciplines within molecular diagnostics, the use of the UNG technology is discouraged; it is only possible to detect contamination from aerosols with amplicons, but not with targets from fresh samples or circulation plasmids.

  7. 7.

    The use of BSA or gelatine is not recommended molecular microbiological analysis; these products might be contaminated by nucleic acids of pathogens [18].

  8. 8.

    Some discussion about terminology; not everybody uses the terminology of Bustin (Bustin SA qPCR A-Z. IUL Biotechnology, No. 5 ISBN-13: 978-0963681782). For instance, the term internal standard is used as an alternative to internal amplification control.

  9. 9.

    In the literature, this negative effect on PCR is defined as inhibition; in fact, the Tm is probably affected in such a way that targets do not melt out correctly. However, interaction with the active centre of the enzyme leading to a real inhibition of the enzyme is also possible. In fact, other intercalating dyes lack such effect.

  10. 10.

    For example, the proofreading enzyme Phusion® Hot Start High-Fidelity DNA Polymerase is blocked by Affibodies® (activated at 98 °C), and AptaTaq® (Roche).

  11. 11.

    Foroota A. Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR); http://dx.doi.org/10.1016/j.bdq.2017.04.001.

  12. 12.

    dPCR is solely available at commercial platforms; may good illustrations can be found by the manufacturers’ Websites.

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Author information

Authors and Affiliations

  1. Leiden Centre for Applied Science, Faculty of Science & Technology, UAC Leiden, Box 382, 2300 AJ, Leiden, The Netherlands

    E. van Pelt-Verkuil

  2. NMDL-LCPL, Visseringlaan 25, 2288 ER, Rijswijk, The Netherlands

    R. te Witt

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  1. E. van Pelt-Verkuil
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Correspondence to E. van Pelt-Verkuil .

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Editors and Affiliations

  1. Leiden Centre for Applied Science, Faculty of Science & Technology, University of Applied Sciences Leiden, Leiden, The Netherlands

    E. van Pelt-Verkuil

  2. Leiden Centre for Applied Science, Faculty of Science & Technology, University of Applied Sciences Leiden, Leiden, The Netherlands

    W.B. van Leeuwen

  3. NMDL-LCPL, Rijswijk, The Netherlands

    R. te Witt

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van Pelt-Verkuil, E., te Witt, R. (2019). Principles of PCR. In: van Pelt-Verkuil, E., van Leeuwen, W., te Witt, R. (eds) Molecular Diagnostics. Springer, Singapore. https://doi.org/10.1007/978-981-13-1604-3_5

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