DNA sequencing is used to determine the exact sequence of nucleotides in a sample of DNA.

One of the most common methods of DNA sequencing is the Sanger, or dideoxy, technique.

Starting off: the ingredients

The DNA of interest is first multiplied many times using PCR.

Next, heat is used to separate the double-stranded DNA molecules into single strands. Several solutions are added: one contains a suitable enzyme (usually Taq polymerase), the second contains a high concentration of DNA nucleotides (deoxynucleotides, dATP, dGTP, dTTP, dCTP), and a third solution contains dideoxynucleotides (ddATP, ddGTP, ddTTP, ddCTP). You also need to add DNA primers. These are short pieces of DNA that have been specially selected to bind to the DNA of interest. They are needed to get the reaction going.

The next steps: making new DNA

Once the primers have bound, the enzyme makes a new strand of DNA by adding together nucleotides in the reaction mixture. The original DNA chain serves as a template. The new DNA is made by complementary base pairing with the original DNA template. Because all four ordinary DNA nucleotides are present in large amounts, the chain elongation continues normally - until by chance a dideoxynucleotide is added in the place of a normal DNA nucleotide.

The dideoxynucleotides are just like ordinary DNA nucleotides except that one OH group has been chemically changed to an H (there is one less oxygen). With normal DNA nucleotides, one nucleotide can be attached to another and so on, forming a chain. The chemical change in a dideoxynucleotide, however, means that no other nucleotides can be added to it.

The animation, Dideoxynucleotides in action was developed by the University of Wisconsin, and shows how the dideoxynucleotides prevent the DNA elongation.

If there are many more ordinary nucleotides than dideoxynucleotides, some chains will be several hundred nucleotides long before a dideoxynucleotide is added. The end result is a whole lot of new DNA chains, of varying length, all ending with a dideoxynucleotide.

Separating and analysing the new DNA strands

The newly made DNA strands can be separated according to their size by gel electrophoresis, which is done in a genetic analyser.

The dideoxynucleotides at the end of each chain are also attached to a dye which fluoresces a particular colour when passed through a laser light beam. A laser light at the end of the gel can therefore be used to make the strands fluoresce as they pass the laser. The particular colour (green, yellow, red or blue) emitted by the dideoxynucleotide at the end of the chain is recorded. Each represents a different nucleotide (A, T, C or G). In this way, the order of nucleotides in the DNA sequence can be obtained.

Why is a DNA sequence useful?

The DNA sequence can be used for a range of different purposes, including identifying regions of variation associated with increased susceptibility to particular diseases (see example). But be careful: Knowing the DNA sequence does not necessarily mean that you know how the DNA is used in the cell.

Published 20 November 2007