Polymerase Chain Reaction
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Figure 2: Thermocycles in PCR reaction. (1)Template denatures to single-stranded DNA. (2) Primers anneal to the templates. (3) DNA synthesis.
Table 1: Number of target DNA molecules produced after n thermocycles.
The exponential phase lasts for approximately 30 cycles under standard reaction conditions. The plateau phase of amplification (Figure 4) is acheived whereby additional thermocycles do not result in significant increase of the amplification products. This is because there are few different factors controlling the exponential and plateau phase amplification.
The exponential phase is only for first 30 cycles, thereby reaching stationary phase. Amplification reaches plateau when one of the PCR components is limiting.
According to Kainz (2000), these factors are:
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DNA synthesis becomes less efficient as the activity of the thermostable DNA polymerase is reduced (due to thermal inactivation, or the availability of DNA polymerase is low that cannot react with exceeding number of amplified target molecules),
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Availability of dNTPs for DNA synthesis is limiting in later cycles,
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Competition between complete amplimers (amplification product) and DNA primers for annealing at target sites,
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Gradual destruction of dNTPs and DNA primers physically,
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Non-specific binding of amplification products to thermostable DNA polymerase substrate binging pocket resulting reduced interations between DNA polymerase and primer/template hyrids,
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Inhibitory effect on DNA synthesis due to accumulation of amplimers and pyprophosphate (PPi) liberated during dNTP hydrolysis.
Due to these factors, it is advisable to stop amplification before the end of the exponential phase is reached as this will help to avoid accumulation of incomplete and non-specific PCR products.
Figure 4: Generation of templates (amplification products) in 0-50 thermocycles yields a S-shaped graph. The exponential phase is only for first 30 cycles, thereby reaching stationary phase. Amplification reaches plateau when one of the PCR components is limiting.
The Thermocycles
The thermocycles of PCR reaction can be simplied as shown in Figure 2. The strength of PCR is that the products resulting from one primer extension are the templates for the next cycle. Consequently, amount of target DNA is doubled after each cycle. The target DNA produced after n cycles of PCR is 2^n whereby the PCR target molecules accumulate as a function of cycle number. Table 1 shows the number of copies after n cycles of PCR reaction.
