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Cycle Sequencing - The Chain Termination Method

dna strand template base

The most popular approach to sequencing is the chain termination method, developed in 1977 by Fred Sanger. This technique makes use of a DNA synthesis reaction and a unique form of a base, called a dideoxynucleotide, that lacks the 3′ hydroxyl chemical group involved in forming the link between nucleotides in a DNA chain. A dideoxynucleotide can be added to a growing chain, but, once incorporated, no further nucleotides can be linked to it. Thus, chain growth is terminated.

In a reaction, the concentration of dideoxynucleotides is optimized so that all possible chain lengths are generated. In automated DNA sequencing, the newly formed chain fragments are marked with four fluorescent dyes, each of which corresponds to one of the four DNA nucleotides (A, C, T, and G). The fragments are then separated by a technique known as gel electrophoresis, during which the dyes are excited and detected by the automated DNA sequencing instrument. In this way, the identity of each successive terminating nucleotide is determined, revealing the sequence of the entire chain.

In a sequencing reaction, all the components needed for the synthesis of DNA are present. These include the DNA to be sequenced (the template) and a short (17 to 28 bases in length), single-stranded piece of DNA (the primer), which attaches itself to a specific site on the template and acts as a starting point for the synthesis of a new DNA strand.

In both a DNA synthesis reaction and in the chain termination method of DNA sequencing, one strand of the template DNA is copied to form a new, complementary strand. An A base on the template strand, for example, directs the addition of its complementary base T into the growing chain. Likewise, a C base on the template strand directs the addition of its complementary base G into the corresponding position of the new chain. During chain growth, an enzyme called DNA polymerase links one nucleotide to the next, extending the new strand until it either reaches the end of the template strand, or, in DNA sequencing, until a dideoxynucleotide is incorporated.

Cycle Sequencing - The Cycle Sequencing Technique [next]

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