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Hydrogen bond

An intermolecular attractive force between hydrogen, when it is covalently bonded to a highly electronegative atom (fluorine, oxygen or nitrogen), and an oxygen, nitrogen or fluorine atom on another molecule.

DNA polymerase

An enzyme involved in the production of a new nucleotide strand to form a new DNA double helix.

Nucleotide

Monomer unit of the nucleic acids DNA and RNA. Each nucleotide is made up of three parts: a pentose sugar, a phosphate group and a nitrogenous base.

Bacteria

Single-celled organism. Has a cell wall, cell membrane, cytoplasm. Its DNA is loosely-coiled in the cytoplasm and there is no distinct nucleus

Cytosine (C)

The nitrogenous base, cytosine, which pairs with G, guanine.

Guanine (G)

The nitrogenous base, guanine, which pairs with C, cytosine.

Nucleus

The part of a cell that controls the cell function and contains the chromosomes.

Thymine (T)

The nitrogenous base, thymine, which pairs with A, adenine.

Adenine (A)

The nitrogenous base, adenine, which pairs with T, thymine.

Enzyme

Reusable protein molecules which act as biological catalysts, changing the rate of chemical reactions in the body without being affected themselves

Bases

Bases, sometimes called nitrogenous bases, are the parts of the DNA molecule that join the two helix strands. They are like rungs on a ladder. There are four bases: adenine (A), thymine (T), guanine (G) and cytosine (C). Each base can only join with one other base; i.e. they join together in pairs: A with T and G with C.

Cell

The basic unit from which all living organisms are built up, consisting of a cell membrane surrounding cytoplasm and a nucleus.

What is the polymerase chain reaction?

DNA is the molecule which carries all our inherited information. It has a double helix structure, and is made up of four nucleotide bases – adenine, thymine, cytosine and guanine - joined together in pairs. Since the structure and importance of DNA was first recognised over sixty years ago the molecule has been studied by thousands of scientists. One development which has made it much easier for everyone working in the field is the polymerase chain reaction (PCR).

PCR has revolutionised molecular biology and DNA technology. Invented by Kary B Mullis, it enables the production of large quantities of DNA from very small samples in a remarkably short time. This in turn makes it possible for us to analyse tiny samples of DNA and unravel the mysteries of individual genes.

Every time a cell in the body reproduces itself, the DNA in the nucleus is copied. The double helix of the DNA ‘unzips’, and the enzyme DNA polymerase makes a copy using the separated strands as templates. For the process to work there must be plenty of nucleotide bases, the small primer sequences which are needed to get the copying process started and the enzyme DNA polymerase.

In 1983 Kary Mullis came up with the idea of using enzymes from a bacterium which lives in the hot springs in Yellowstone National Park to develop a technique for replicating DNA artificially in the lab. His idea worked, and he won a Nobel prize in 1993.

Extremophiles from hot springs are the key to the polymerase chain reaction.
(Jon Sullivan)

How does PCR work?

The polymerase chain reaction depends on the fact that the different stages of DNA replication, using enzymes from Thermus aquaticus, take place at different temperatures. So by mixing all the ingredients together at the beginning and then changing the temperature of the mixture in the PCR machine a tiny amount of DNA can be amplified to produce millions of identical molecules.

The stages of the process

Stage 1:The reactants are mixed together in a PCR vial. The mixture contains the DNA which is to be amplified, the enzyme DNA polymerase, small primer sequences of DNA and a good supply of the four nucleotide bases A,T,C and G. The vial is placed in a PCR machine.

PCR Stage 1

Stage 2: The reaction mixture is heated to 90-95oC for about thirty seconds. At this temperature the DNA strands separate as the hydrogen bonds holding them together break down.

PCR Stage 2

Stage 3: The mixture is cooled down to 50-60oC. At this temperature the primers bind (or anneal) to the single DNA strands. The primers are short sequences of nucleotide bases which must join to the beginning of the separated DNA strands for the full copying process to start.

PCR Stage 3

Stage 4: In the final step the mixture is heated up again to 75oC for at least a minute. This is the optimum temperature for the DNA polymerase enzyme. The enzyme adds bases to the primer segments to build up complementary strands of DNA identical to the original molecule.

PCR Stage 4

These last three steps can be repeated around thirty times to give around 1 billion copies of the original DNA. The whole process takes only about 3 hours – and much of that is the time taken heating and cooling the reaction mixture in the PCR machine.

PCR animation

Click on the play button to start the animation. Use the other buttons to stop the animation at any point, to skip to the end of the animation or to return to the start.