Biotechnology Basics: An Introduction to PCR

What is the polymerase chain reaction (PCR)? PCR is a technique that allowsresearchers to quickly create many copies of a specific region of DNA in vitro.

What do I need to perform PCR?

• Template – the purified, double-stranded piece of DNA we want to copy
• Primers – short synthetic DNA molecules that target a specific DNA sequence for amplification
• Taq DNA Polymerase – thermostable enzyme used to copy DNA
• Free nucleotides – the building blocks of DNA
• Thermal Cycler (a.k.a. PCR machine) – a specialized machine that rapidly heats and cools the samples.

How does PCR work?

To perform PCR, the template is mixed with primers, Taq polymerase and nucleotides. Themixture is heated to 94°C to denature the DNA duplex (i.e., unzip it into single strands).  Next, the sample is then cooled to 45°C-60°C, allowing the primers to base pair with the target DNA sequence (called “annealing”).  Lastly, the temperature is raised to 72°C, the optimal temperature at which Taq polymerase will extend the primer to synthesize a new strand of DNA. Each “PCR cycle” (denaturation, annealing, extension) doubles the amount of the target sequence in less than five minutes.  In order to produce enough DNA for analysis, twenty to forty cycles may be required.

What are some applications of PCR?

Because of its ease of use and its ability to rapidly amplify DNA, PCR has become indispensible in medical and life sciences labs. For example, research laboratories can amplify a specific region of DNA for cloning applications. Medical tests use PCR to identify genetic mutations and infectious agents.  In addition, because amplification by PCR requires very little starting material, it is ideal for forensic analysis of biological samples.

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