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What Is Polymerase Chain Reaction - PCR

Polymerase Chain Reaction - PCR - Defined

PCR is a technique that is used to rapidly create multiple copies of a segments of DNA using repeated cycles of DNA synthesis. PCR has revolutionized molecular biology and forensics, allowing amplification of small quantities of DNA into amounts that can be used for experimentation or for forensic testing.

From trace amounts of the DNA used as starting material (template), PCR produces exponentially larger amounts of a specific piece of DNA. The template can be any form of DNA, and only a single molecule of DNA is needed to generate millions of copies. PCR makes use of two normal cellular activities:

  1. Binding of complementary strands of DNA, and
  2. replication of DNA molecules by DNA polymerases

PCR DNA Amplification

How DNA gets amplified in a PCR reaction

How DNA gets amplified in a PCR reaction

A Thermal Cycler

A thermal cycler or thermocycler allows a PCR reaction to occur

A thermal cycler or thermocycler allows a PCR reaction to occur

Who Invented PCR?

Kary Mullis together with Michael Smith came up with the technique of PCR in 1983. A Nobel Prize was awarded in half to Kary Mullis and half to Michael Smith in 1993 for their invention of the technique.

Thermal cyclers were only invented later. Before the automated process,water baths set at various temperatures were used and researchers timed the length of time reaction tubes stayed in one bath before being moved to the next water bath at a different temperature.

What Happens in a PCR Reaction?

A few simple things happen quickly in a PCR reaction.

  1. Denaturing - at 95 degrees celsius the single stranded template DNA is broken apart
  2. Annealing - from 45-68 degrees celsius the primers attach to the homologous DNA sequence that they match
  3. Extension - Taq polymerase begins adding nucleotides around 72 degrees celsius and extending the DNA sequence.

The cycles repeat around 30-40 times depending on how much DNA is desired and in the end in each cycle the DNA doubles 2,4,8,16,32,64,128,256…and so on.

Programming a PCR Reaction

An example of a polymerase chain reaction heating curve. Individual temperatures may vary based on individual experiments.

An example of a polymerase chain reaction heating curve. Individual temperatures may vary based on individual experiments.

A nucleotide, thymine, in a PCR reaction

A nucleotide, thymine, in a PCR reaction

Ingredients for a PCR Reaction

The following reactants are needed to successfully carry out a PCR reaction.

  • Taq Polymerase
  • MgCl2 - buffer (optional)
  • deoxyribonucleotides (dntp's): Adenine, Thymine, Guanine, Cytosine
  • Water
  • DNA Template
  • Forward primer - specific tor your DNA template
  • Reverse primer - specific to your DNA template

Designing Primers

  • Primers must be at least 20 base pairs long.

Shorter primers will not allow Taq polyymerase to adhere properly before the extension step, consequently base pairs cannot be added and DNA synthesis will not proceed.

  • Primers must match target DNA sequences.


Using GenBank homologous sequences can be found from evolutionary related organisms if the specific sequence of your target DNA has not bee discovered already. The more specific the primers are to the target DNA sequence the more likely they are to bind to that correct sequence and amplify only that sequence. Less specific primers bind with less precision and have a chance of amplifying the incorrect target DNA.

Taq Polymerase Structure

Taq Polymerase Structure

Taq Polymerase Structure

What is Taq Polymerase?

Taq polymerase is an enzyme that adds nucleotides to the growing DNA chain.

How does Taq Polymerase differ from regular DNA polymerase? Is like DNA Polymerase, except more heat stable “thermostable”. This means that at 95 degrees Celsius when the DNA is being opened apart, the enzyme will not denature and lose function in subsequent steps of the PCR reaction.

Taq polymerase was isolated from Thermus aquaticus, a bacteria that live in hot springs in Yellowstone National Park. Since the hot springs frequently approach boiling temperatures, T. aquaticus and other bacterial species that live in these waters must have enzymes that are functional at high temperatures, so the DNA polymerase is not inactivated by the denaturation step.

Programming a PCR Cycle

How to set up a PCR reaction program

CycleTemperatureTime

Initial denaturation x1

95 C

30 sec

Thermocycling x30

95 C

15-30 sec

 

45 - 68 C

15-60 sec

68 C

1 min per kB

Final Extension

68 C

5 minutes

Hold

4 C

Overnight

Example of a Real PCR Program Cycle

A PCR Reaction Program Setup

A PCR Reaction Program Setup

rt-PCR Reverse-transcriptase PCR

Reverse-transcriptase PCR or real-time PCR you can track the amplification of your DNA.using transmittance or absorbance data, to quantify how active a gene is in a sample.

For example, you can isolate the RNA of a protein, use rt-PCR to make DNA and quantify using a fluorescent tag, how much of that initial RNA you have and how much is at the end of your thermocycles.

rt-PCR Amplification Curves

rt-PCR amplification curves of DNA

rt-PCR amplification curves of DNA

Applications of PCR

  • Genetic testing - detect mutations
  • Forensics - genetic fingerprinting
  • Detecting oncogene mutations in cancer
  • HIV - detect 1 viral genome replicating among 50,000 host cells
  • Building phylogenetic trees
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