DNA sequencing is the process of determining the order of nucleotides on a segment of DNA. Cycle sequencing is a method used to increase the sensitivity of the DNA sequencing process and permits the use of very small amounts of DNA starting material. This is accomplished by using a temperature cycling process similar to that employed in the polymerase chain reaction.
The Chain Termination Method
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.
The Cycle Sequencing Technique
In cycle sequencing, a reaction is taken through several steps designed to prepare the template for copying, allow for initiation of DNA synthesis, and generate the terminated DNA chains needed for electrophoresis and sequence determination. The first step in the process is called denaturation, in which double-stranded template DNA is converted into its single-stranded form. This is accomplished by heating the template to between 94 °C and 98 °C, a temperature high enough to break the hydrogen bonds between the complementary bases holding the two strands together.
As a single-stranded molecule, the template's bases are now exposed, and are free to interact with the sequencing primer. The primer, in a step called annealing, locates and attaches itself to its complementary site on the template. Thus, Ts bind to As and Cs bind to Gs. However, primer annealing will only occur at a temperature where hydrogen bonds can form between the primer and template strands, usually between 40 °C and 65 °C. Because the high temperature used for the denaturation step in each cycle would destroy most DNA polymerase enzymes, a special heat-stable enzyme must be used in the annealing stage, one that remains active even after repeated exposure to very high temperatures. The enzyme most commonly used at this point in cycle sequencing is Taq, isolated from Thermus aquaticus, a bacterium that lives in the hot springs of Yellowstone National Park.
In the final step of the reaction, DNA polymerase extends the annealed primer by sequentially adding on to its end bases that are complementary to those on the template. It is during this extension step of a DNA sequencing reaction that random incorporation of a dideoxynucleotide can occur, terminating chain growth. All three of these steps, taken together, represent one round, or cycle, of a DNA synthesis reaction. By repeating a cycle over and over again, the amount of each fragment made in the reaction can be substantially increased. Since each fragment carries fluorescent dyes, increasing the number of copied fragments also increases the strength of the fluorescent signal. Cycle sequencing, therefore, greatly improves the sensitivity of the sequencing reaction, and even very small amounts of starting DNA sample can be used as template.
see also Automated Sequencer; Gel Electrophoresis; Polymerase Chain Reaction; Sanger, Fred; Sequencing DNA.
Frank H. Stephenson
and Maria Cristina Abilock
Craxton, Molly. "Linear Amplification Sequencing, a Powerful Method for Sequencing DNA." In METHODS: A Companion to Methods in Enzymology 3 (1991): 20-26.