Automated Sequencing With Fluorescent Dyes
As originally developed, both the Maxam-Gilbert and the chain termination methods of DNA sequencing require the use of radioisotopes to visualize the fragments generated in the reactions. However, in addition to being a health risk and presenting a disposal problem, the use of radioactivity makes automation of the DNA sequencing process difficult. Machines that could automatically read DNA sequences did not become practical until fluorescent dyes were introduced as a way to label sequencing fragments.
Two approaches are used to fluorescently label the products of a DNA sequencing reaction: the dye primer method and the dye terminator method. Both are applied only to the chain termination method. The sequencing products made by these methods are electrophoresed on an instrument that uses a laser to detect the different fragments.
In the dye primer method, fluorescent dyes are attached to the 5′ ("five prime") end of the primer, which is the end opposite to that where nucleotides are added during chain growth. Four reactions are prepared containing all the components described above, but with no radioisotopes. In the "A" reaction, the primer carries a dye at one end that fluoresces green when struck with a laser. In the "C" reaction, the primer carries a dye that fluoresces blue. The "G" reaction's primer has a yellow dye on its end and the "T" reaction's primer carries a red dye.
Termination events in the "A" reaction tube result in fragments with a green dye at one end and a dideoxyadenosine at the other. Termination events in the "C" reaction result in the formation of fragments having a blue dye at one end and a dideoxycytidine at the other. Similar relationships hold in the "G" and "T" reactions. When incubation is complete, the four reactions are combined and electrophoresed in a single gel lane of an automated sequencer. A laser, shining at the bottom of the gel, excites the dyes on the DNA fragments, causing them to fluoresce as they pass. The instrument's optics system detects the fluorescent colors during electrophoresis and a computer then translates the order of colors into a base sequence (see Figure 3).
In the second approach to fluorescent DNA sequencing, the dye terminator method, dyes are attached to the dideoxynucleotides instead of to the primers. The DNA fragment in the sequencing reaction becomes dye-labeled when a dideoxynucleotide is incorporated. The dye terminator method uses a single reaction tube (rather than four) because each dideoxynucleotide is associated with a different dye. The cost and time for sequencing are therefore reduced, making this approach the preferred method used by most laboratories.
Manufacturers of DNA sequencing reagents now provide kits that contain all the components necessary for a sequencing reaction in a "master mix" format. For the dye terminator approach, a master mix can be purchased that contains DNA polymerase, the four nucleotides, buffer, magnesium, and the four dye-labeled dideoxynucleotides. The addition of template and primer to the master mix completes the reaction.
In addition to the widespread use of sequencing kits, other improvements have been made. Enhanced signal strength and improved sensitivity have been achieved through the development of stronger fluorescent dyes and the exploitation of heat-stable DNA polymerases that allow for repeated cycling of the sequencing reaction.
Frank H. Stephenson
and Maria Cristina Abilock
Bloom, Mark V., et al. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. San Francisco: Benjamin Cummings Publishing, 1995.
Maxam, Allan M., and Walter Gilbert. "A New Method for Sequencing DNA." Proceedings of the National Academy of Sciences 74 (1977): 560-564.
Sanger, Frederick, S. Nicklen, and Alan R. Coulson. "DNA Sequencing with Chain-terminating Inhibitors." Proceedings of the National Academy of Sciences 74 (1977): 5463-5467.