History of Chain termination method

Difference (from prior major revision) (minor diff, author diff)

Changed: 1c1

The Chain termination or Sanger or Dideoxy method is a process used to sequence (read the bases) of DNA. It is named after [Fred Sanger]? who developed the process in 1965.

The Chain termination or Sanger or Dideoxy method is a process used to sequence (read the bases) of DNA. It is named after Frederic Sanger who developed the process in 1965.

Changed: 3,6c3,6

The process relies restriction enzymes to produce DNA of varying lengths. On cloned pieces of DNA the enzymes stop DNA replication at one of the four bases. The lengths of the DNA strands can be worked out using gel electrophoresis.

Process

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. Each of these floresces a different colour when illuminated by a laser beam. The enzyme [DNA polymerase]? is then added.

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.

Detailed process

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.

Changed: 10c10,13

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

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 diplacement from the original primer.

Uses

The dideoxy method is now highly automated as a result of development occuring under the Human Genome Project, where it is used to sequence fragments of our genome.

Changed: 12,13c15

Uses

The dideoxy method is now highly automated as a result of development occuring under the Human Genome Project, where it is used to sequence fragments of our genomes.

/Talk?

	Revision 7 . . (edit) November 26, 2001 10:17 pm by Sodium
	Revision 6 . . (edit) November 26, 2001 10:17 pm by Sodium [wording]
	Revision 5 . . (edit) November 26, 2001 9:17 pm by Malcolm Farmer
	Revision 4 . . (edit) November 24, 2001 1:23 am by Sodium
	Revision 3 . . (edit) November 19, 2001 5:55 am by Sodium
	Revision 2 . . November 19, 2001 4:28 am by Larry Sanger [Changing [[Fred Sanger]] to [[Frederic Sanger]], which exists. Please search for existing articles when creating links!]
	Revision 1 . . November 19, 2001 2:30 am by Sodium [initial article]