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Chain termination method
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.
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.
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.
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.
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