DNA sequencing mimics the basic process used to copy DNA in a cell during chromosomal replication, except that the procedure is done in a tube or microtiter plate using a minimal set of components. Most DNA sequencing techniques require that there be a "template", (i.e., a biological sample of the DNA whose sequence is to be determined); a "primer", (i.e., a short oligonucleotide that is complementary to a region of the template and capable of being extended); and a DNA polymerase enzyme that successively adds building blocks on to a primer, as directed by the template strand; and the four building blocks themselves. The technique also must embody a method by which the order of the building blocks added to the primer can be detected. Using the detection method of choice, the sequence of the DNA strand complementary to the template is, thereby, determined.
Most large-scale DNA sequencing facilities use fluorescent dyes to label and detect the four bases, and capillary electrophoresis to separate DNA molecules on the basis of size so that the base located at each position in the sequence can be identified. More specifically, for a small percentage of the molecules of each building block added to the sequencing reaction, the building block is chemically modified and labeled with a distinguishable dye such that when a modified building block is randomly added to the DNA strand being extended from the primer, the replication "terminates", with the result that the sequencing reaction contains a mixture of molecules of varying sizes. Because the end of each terminated molecule contains a dye-labeled base, the sequence of the strand complementary to the template can be determined.
The image above shows a set of sequencing lanes, where electrophoresis is used to separate molecules differing by one base. Laser detection is used to identify the bases at each position. The sequence is "read" from the bottom up, using a key where "A" is green, "C" is blue, "G" is yellow, and "T" is red. Using software provided by the manufacturers of sequencing machines, the signal/noise ratios of the dyes is determined for each position so that the proper base can be "called". The order of the bases is displayed in a "chromatogram" or "trace" file.
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