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Chapter 2.3.2 : PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification

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PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, Page 1 of 2

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Abstract:

The field of molecular biology was revolutionized with the development of the polymerase chain reaction (PCR). This chapter defines PCR, reverse transcription PCR (RT-PCR), real-time PCR, digital PCR and isothermal amplification. Within each subject a brief overview of the process is given along with the required reagents or components and highlighted applications. RT-PCR allows detection and characterization of RNA with options for one-step and two-step RT-PCR procedures with different advantages and disadvantages. Real-time PCR is typically coupled with a fluorescent-based reporter system such as an intercalating dye or a sequence specific probe. Unlike conventional PCR, real-time PCR can be used to quantity the amount of nucleic acid in a given sample where absolute quantification requires the use of known standards to calculate the concentration for a sample, while relative quantification uses a "calibrant" to determine the fold change in a sample. Real-time PCR can be used for direct measurement of DNA targets or it can be coupled with RT-PCR to quantify RNA targets. Digital PCR has only recently become widely available and provides a means to quantify targets in a sample based on direct estimation rather than by making estimates from standard curves. Many isothermal amplification methods have been developed to amplify nucleic acid targets without the need for thermalcycler technologies.

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2
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FIGURE 1

Principles of PCR. doi:10.1128/9781555818821.ch2.3.2.f1

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2
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Image of FIGURE 2
FIGURE 2

Oligonucleotide specific probes: hydrolysis probes (a) and molecular beacons (b). doi:10.1128/9781555818821.ch2.3.2.f2

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2
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Image of FIGURE 3
FIGURE 3

Typical plot of a real-time PCR reaction. (1) Plateau phase where critical reactants are exhausted, (2) late exponential/linear phase where reactants become limited, (3) exponential phase, and (4) pre-exponential phase. doi:10.1128/9781555818821.ch2.3.2.f3

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2
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Image of FIGURE 4
FIGURE 4

Calculation of the standard curve for absolute quantification. Dilutions of known concentration are run (a), and the values are plotted (b) as a function of quantity (x axis, logarithmic) and value (y axis). doi:10.1128/9781555818821.ch2.3.2.f4

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2
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Image of FIGURE 5
FIGURE 5

Delta-delta relative PCR quantitation method. A basic overview of the ΔΔ is shown. The overall difference in between expression within (Δ) and between (ΔΔ) is shown above. In this example, the calibrant/reference gene is compared to the target gene of interest in the control (a) and the experimental (b) groups. doi:10.1128/9781555818821.ch2.3.2.f5

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2
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Tables

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TABLE 1

Isothermal amplification methods and reviews

Citation: Bartholomew R, Hutchison J, Straub T, Call D. 2016. PCR, Real-Time PCR, Digital PCR, and Isothermal Amplification, p 2.3.2-1-2.3.2-13. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.3.2

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