Cycle Sequencing

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.