In 1974, Sanger developed a method of DNA sequencing based on the principles of DNA replication.  In this process, nucleotide- based analogs, which lack the 3’-hydroxyl group (also named as dideoxynucleotides) are mixed with other dNTPs for DNA replication.  The analogs are incorporated into DNA replication by DNA polymerase, and, once they are incorporated, DNA extension stops.  In a traditional experimental set up, four reaction tubes are prepared, each containing radioactively- labeled nucleotides and either ddA, ddC, ddG, or ddT.  During DNA extension, the radioactive dideoxynucleotide (ddA, ddC, ddG, or ddT) is added to the 3’ end and terminates the extension.  When the reaction completes in each reaction tube, extension products of various lengths are produced with the same dideoxynucleotide at  the 3’end.  The extension products are then separated by polyacrylamide gel electrophoresis.  The separation has a resolution of one base.  The gel image is then used to determine the sequence.

The first generation automated sequencing technology is “cycling sequencing”, which is still based on Sanger chemistry.  It includes dye primer and dye terminator chemistry.

For dye primer chemistry, the reaction setup is very similar to Sanger DNA sequencing.  As shown above, four primers, labeled with different dyes, are separated into four reactions; in each reaction, a dideoxynucleotide ,that correspondents to the dye, is added.  The reactions are carried out in a thermal cycler device with thermal stable Taq DNA polymerase.  When the PCR cycle completes, each reaction will contain various lengths of PCR products that are terminated randomly by dideoxynucleotide, but labeled by one specific dye.  The products from four reactions will be combined and electrophoresed through the capillary electrophoresis instrument. 

The dye terminator chemistry is a single-tube reaction that uses only one unlabeled primer, plus  a template, a dNTP (labeled ddNTP), a Taq DNA polymerase, and a buffer. The four ddNTP are labeled with different dyes.  Various lengths of DNA fragments are generated during the PCR cycles when ddNTP is incorporated to the end of the sequence, as shown below.   The end products are analyzed through capillary electrophoresis.

The end products ,from either dye primer or dye terminator chemistry, are separated by their sizes in the electrophoresis field.  Capillary electrophoresis can resolve DNA molecules that differ in molecular weight by only one nucleotide.  Before different fragments migrate through capillaries to reach positive electrodes, a laser beam excites the fluorescent dye and an emitted fluorescent light from individual fragments is detected by a fluorescence detector.  The instrument determines the DNA sequence by the dyes that pass through a laser beam.