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DNA sequencing

Sequencing is a technique used for "reading" the precise order of nucleotides in a DNA fragment. Small DNA fragments, whole genes, or even genomes can be sequenced.

The most widely used DNA sequencing technique is based on chain-termination developed by Sanger. This method is very similar to polymerase chain reaction (PCR) but involves only one primer, which anneals close to the region of interest at the 3' end of the DNA template (Figure 1). During the sequencing reaction a mixture of normal nucleotides (dATP, dTTP, dCTP, and, dGTP) and "stop"-nucleotides (ddATP, ddTTP, ddCTP, and ddGTP) are added to the DNA template. The stop nucleotides are each labeled with a specific color. In every cycle the target DNA is replicated by a DNA polymerase until a stop-nucleotide is added, which stops further elongation (chain-termination). After 35 cycles, a large number of fragments in all possible lengths are produced. These fragments are run in a specialized acrylamide gel where their length and "end-bases" are detected. Because the fragments are separated based on their size in the gel, the labeled nucleotides are detected one by one and thus the precise DNA sequence in the fragment can be reconstructed.

The purpose of sequencing is, for example, to predict the protein sequence of a gene, to compare species on a sequence level (genes or genome), or to search for a mutation.

Top left corner of an image presents four spheres of different colors with four different letters inside of them, called Fluorescently labeled ddNTPs. Arrow down points toward a DNA template consisting of blue spheres in a row with four different letters inside. Another arrow points down toward rows of DNA sequences, each row has one base less than the upper one, with the last base on the left filled with a color, matching the colors of ddNTPs above. From these rows, one arrow points right to the grey, horizontal rectangle with four wells, each for one DNA base, and colored bands of various sizes, matching colors of ddNTPs. Another arrow from the rows points at the bottom right part of an image, showing a graph, where each peak indicates one DNA base.

Figure 1: Schematic overview of Sanger (chain-termination) method for DNA sequencing

DNA fragments of all possible lengths are produced during the elongation cycles, each terminated with a colored stop nucleotide. When the fragments are separated according to their size on a gel, the order of colors detected will indicate the precise DNA sequence.

Referred from:

Article
Gene expression
Article
Nucleic acid-based assays