Chain termination method

From Academic Kids

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. Variations on the classical Sanger method have led to the development of an automated sequencing method.

In the chain termination method, the DNA segment to be sequenced is replicated over and over. Dideoxynucleotides 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.

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 this way. Therefore longer segments of DNA are sequenced using methods (such as chromosome walking and shotgun sequencing) which sequence multiple short strands in such a way that they can be "assembled" to reveal the sequence of a much longer strand.

Detailed Sanger Method

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Sequencing.jpg
The Sanger Method (Classical DNA sequencing)

The classical chain termination method or Sanger method first involves preparing the DNA to be sequenced as a single strand. The DNA sample is divided into four separate samples. Each of the four samples have a primer, the four normal deoxynucleotides (dATP, dGTP, dCTP and dTTP), DNA polymerase, and only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP and ddTTP) added to it. The dideoxynucleotides are added in limited quantities. The primer or the dideoxynucleotides are either radiolabeled or have a fluorescent tag.

As the DNA strand is elongated the DNA polymerase catalyses the joining of deoxynucleotides to the corresponding bases. However, if a dideoxynucleotide is joined to a base, then that fragment of DNA can no longer be elongated since a dideoxynucleotide lacks a crucial 3'-OH group. 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.

The DNA is then denatured and the resulting fragments are separated (with a resolution of just one nucleotide) by gel electrophoresis, from longest to shortest. Each of the four DNA samples is run on one of four individual lanes (lanes A, T, G, C) depending on which dideoxynucleotide was added. Depending on the whether the primers or dideoxynucleotides were radiolabeled or fluorescently labeled, the DNA bands can be detected by exposure to X-rays or UV-light and the DNA sequence can be directly read off the gel. In the image on the right, X-ray film was exposed to the dried gel, and the dark bands indicate the positions of the 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.

Detailed Automated Sequencing Method

Advances in science have led to the development of automated sequencing, a variation on the classical Sanger method. Automated sequencing employs cycle sequencing. In this method thermostable DNA polymerase, the four normal deoxynucleotides, the four dideoxynucleotides (added in limited quantities), and a primer are added to the DNA sample to be sequenced. Unlike the Sanger method, there is no need to divide the DNA into four samples. Each type of dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP) is labeled with a different colored fluorescent dye. In a variation on PCR, only one primer is used and thus amplification is linear. When the reaction is complete the DNA is denatured and separated by gel electrophoresis like in the Sanger method. Since each type of dideoxynucleotide is fluorescently labeled with a different color a laser can read the gel and determine the DNA sequence.

Uses

The dideoxy method became highly automated due to development during the Human Genome Project, where it was used to sequence fragments of our genome.

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