The chain termination or Sanger or dideoxy method is a process used to sequence (read the bases of) DNA. It is named after Frederick Sanger who developed the process in 1975. For the sense of sequencing used in electronic music, see the music sequencer article. ... Space-filling model of a section of DNA molecule Deoxyribose nucleic acid (DNA) is a nucleic acid that contains the genetic instructions specifying the biological development of all cellular forms of life (and many viruses). ... This article or section should be merged with Fred Sanger Frederick Sanger OM (born August 13, 1918) is a British molecular biologist who was working on problems related to the determination of the structure of proteins. ... 1975 was a common year starting on Wednesday (the link is to a full 1975 calendar). ...

The polymerase chain reaction is used to produce millions of cloned pieces of DNA. Substances are added to randomly stop the creation of DNA at each of the four bases (depending on the substance), producing pieces of DNA of almost every length. The lengths of the DNA strands can be worked out using gel electrophoresis. Markers on each strand show which base each strand ends with. When the results from the strands are combined, it is possible to work out the sequence of bases at any point. Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying (creating multiple copies of) DNA without using a living organism, such as E. coli or yeast. ... Cloning is the process of creating an identical copy of an original. ... SDS-PAGE autoradiography Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. ...

Because only one base pair can be read off for each strand produced during the process, only a small section of DNA can be reliably sequenced in this way. More complex sequencing methods, such as chromosome walking and shotgun sequencing, therefore sequence multiple short strands in such a way that they can be "assembled" to reveal the sequence of a much longer strand. Chromosome walking is a method in genetics for identifying and sequencing long parts of a DNA strand, e. ... Shotgun sequencing is a method used in genetics for sequencing long DNA strands. ...

Detailed process

DNA sequencing

A primer is added to a single-strand length of DNA to be sequenced. To this template DNA is added a mixture of the four normal deoxy-nucleotides (dATP, dGTP, dCTP and dTTP). Also added are the dideoxy-nucleotides (ddATP, ddGTP, ddCTP and ddTTP) in limited quantities. These have a fluorescent tagging such that each of these fluoresces a different colour when illuminated by a laser beam. The enzyme DNA polymerase is then added. DNA sequencing gel. ... A primer is a nucleic acid strand (or related molecule) that serves as a starting point for DNA replication. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. ... DNA polymerase 3D structure. ...

The DNA polymerase catalyses the joining of deoxy nucleotides to the corresponding bases. However if by chance a dideoxy nucleotide is joined to a base, then that fragment of DNA can no longer be elongated. Because a dideoxy nucleotide lacks a crucial -OH group, it is impossible to add any other base after a ddNTP. Fragments of all sizes should be obtained due to the randomness of when a dideoxy nucleotide is added. However, to make sure that all different lengths will occur, only short stretches of DNA can be sequenced in one test.

When incubation is complete, the fragments are separated (with a resolution of just one nucleotide) by gel electrophoresis, from longest to shortest. As each tagged dideoxy nucleotide fluoresces a different colour under laser light, it is possible to read off which base occurs at each displacement from the original primer.

For the image shown to the right, four sequencing reactions were performed, each with random chain terminations for one of the four DNA subunits (lanes A, T, G, C). All synthesized DNA was radioactively labeled. Medical X-ray film was exposed to the dried gel, dark bands indicate the positions of the radioactive DNA molecules of different lengths. A dark band in a lane indicates a chain termination for that particular DNA subunit and the DNA sequence can be read off as indicated.

Uses

The dideoxy method is now highly automated as a result of development occurring under the Human Genome Project, where it is used to sequence fragments of our genome. The Human Genome Project (HGP) endeavored to map the human genome down to the nucleotide (or base pair) level and to identify all the genes present in it. ... In biology the genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). ...