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Next-generation sequencing

Next-generation sequencing replaced the traditional Sanger method of DNA sequencing. The technique involves millions of strands of DNA being sequenced at the same time, making the process both faster and cheaper. It is also known as massively parallel sequencing.

Dideoxynucleotide

A specially modified DNA base which attaches to a sequence of bases in a DNA chain and prevents any further elongation of the chain

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.

Cytosine (C)

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

Adenine (A)

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

Guanine (G)

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

Thymine (T)

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

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.

Sequencing the DNA

When DNA sequencing first began, every step was done individually by scientists. The first automated method for sequencing the DNA was the Sanger method, used in the Human Genome Project to sequence the human genome for the very first time. Although genomes are now analysed using even more automated, faster and cheaper next-generation sequencing technologies, these new machines still rely heavily on the basic processes of the Sanger method outlined here.

A flask is set up containing the single stranded DNA which is going to be sequenced along with the enzyme DNA polymerase, an excess of normal nucleotide bases - adenine (A), thymine (T), guanine (G) and cytosine (C) - and a limited number of dideoxynucleotide bases. The addition of a dideoxynucleotide base at the end of a chain stops the addition of any more bases to that end of the chain. Each different type of dideoxynucleotide base (A, T, G and C) has a different coloured fluorescent tag (often four separate flasks are set up, each containing DNA, DNA polymerase and normal bases, but containing only one type of dideoxynucleotide base). Flasks are incubated at the optimum temperature for the reaction.

The DNA polymerase starts to build up new DNA strands based on the single strand template. Each time a dideoxynucleotide base is incorporated, the chain is terminated – no more bases can be added. As a result many DNA fragments are made, all of different lengths as the chain terminating bases are added at random during the process.

The DNA fragments undergo gel electrophoresis which separates them out. The smallest fragments travel the furthest through the gel, with the larger fragments travelling least. The fluorescent markers can then be used to identify the final base on each of the fragments.

Reading the sequence of bases from the electrophoresis plate lets us see the sequence of the new complementary strand of DNA which has been made. This provides all the information needed to determine the sequence of the original DNA.

See an animation of DNA sequencing and try sequencing DNA for yourself.