dyeterminatorsekvensering

Dye terminator sequencing, also known as Sanger sequencing, is a method used to determine the nucleotide sequence of a DNA fragment. It was developed by Frederick Sanger in 1977. The technique relies on the use of dideoxynucleotides (ddNTPs) which, when incorporated into a growing DNA strand, cause premature termination of DNA synthesis.

In a typical dye terminator sequencing reaction, DNA is amplified and then subjected to a chain termination reaction. This reaction involves DNA polymerase, primers, dNTPs, and fluorescently labeled ddNTPs. There are four different fluorescent dyes, each corresponding to one of the four bases (A, T, C, and G). During DNA synthesis, when a ddNTP is incorporated, the chain elongation stops. Each ddNTP is labeled with a unique fluorescent dye, allowing for the identification of the terminal base.

After the chain termination reaction, the resulting DNA fragments, which vary in length by a single nucleotide, are separated by size using capillary electrophoresis. As each fragment passes a detector, the fluorescent dye at its 3' end is excited, and its color is recorded. A laser excites the dyes, and a detector records the emitted fluorescence. The sequence of colors detected corresponds to the sequence of bases in the original DNA fragment. The data is then analyzed by specialized software to generate the DNA sequence. Dye terminator sequencing is a highly accurate method and was the gold standard for DNA sequencing for many years, though it has largely been superseded by next-generation sequencing technologies for large-scale projects.