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DNA Amplification

PCR

The polymerase chain reaction (PCR) is a method to rapidly amplify sequences of DNA. During a typical PCR, template DNA (containing the region of interest) is mixed with deoxynucleotides (dNTPs), a DNA polymerase and primers. Primers are short segments of complimentary DNA that base-pair with the template DNA upstream of the region of interest and serve as recruitment sites for the polymerase. PCR involves a series of temperature cycles that, although once conducted by moving tubes through various water baths, is now controlled automatically by the use of thermal cyclers, or thermocyclers. Thermocyclers provide tight control over both the reaction temperature and the duration of each temperature step, ensuring efficient amplification. During a typical PCR, cycles of denaturation, annealing and extension are repeated to achieve exponential amplification of the target sequence. Denaturation consists of heating the samples up to a high temperature (typically 94-98°C) to cause denaturation of the template DNA, disrupting the hydrogen bonds and base stacking interactions that hold the DNA strands together. 
Once the strands are separated, the temperature is decreased to the annealing temperature to allow the primers to base pair (or anneal) to complimentary regions of the template. The annealing temperature (typically between 48-72°C) is related to the melting temperature (Tm) of the primers and must be determined for each primer pair used in PCR. During the extension step (typically 68-72°C) the polymerase extends the primer to form a nascent DNA strand. This process is repeated multiple times (typically 25-35 cycles). And because each new strand can also serve as a template for the primers, the region of interest is amplified exponentially. The final step of the PCR is generally a longer, single temperature step (often 5-10 min at 68-72°C) that allows for the completion of any partial copies and the clearance of all replication machinery from the nascent DNA. Once the PCR is complete, the thermal cycler is set to 4°-10°C to maintain product integrity until such time as the tubes can be removed from the machine.


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PCR includes these areas of focus:
Routine PCR
High-Fidelity PCR
Protocols for PCR
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Videos

  1. PCROverview_thumb

    Overview of PCR

    This overview will walk you through how the Polymerase Chain Reaction (PCR) works.

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