DNA sequencing

DNA sequencing is a laboratory method used to determine the sequence of a DNA molecule. The method was developed by Frederick Sanger in 1975, who was later awarded the Nobel Prize in chemistry in 1980 for his contributions to understanding DNA sequences. Consequently, it is often referred to as Sanger Sequencing.

In Sanger sequencing, the DNA to be sequenced serves as a template for DNA synthesis. A DNA primer is designed to be a starting point for DNA synthesis on the strand of DNA to be sequenced. Four individual DNA synthesis reactions are performed. The four reactions include normal A, G, C, and T deoxynucleotide triphosphates (dNTPs), and each contains a low level of one of four dideoxynucleotide triphosphates (ddNTPs): ddATP, ddGTP, ddCTP, or ddTTP. The four reactions can be named A, G, C and T, according to which of the four ddNTPs was included. When a ddNTP is incorporated into a chain of nucleotides, synthesis terminates. This is because the ddNTP molecule lacks a 3' hydroxyl group, which is required to form a link with the next nucleotide in the chain. Since the ddNTPs are randomly incorporated, synthesis terminates at many different positions for each reaction.

Following synthesis, the products of the A, G, C, and T reactions are individually loaded into four lanes of a single gel and separated using gel electrophoresis, a method that separates DNA fragments by their sizes. The bands of the gel are detected, and then the sequence is read from the bottom of the gel to the top, including bands in all four lanes. For instance, if the lowest band across all four lanes appears in the A reaction lane, then the first nucleotide in the sequence is A. Then if the next band from bottom to top appears in the T lane, the second nucleotide in the sequence is T, and so on. Due to the use of dideoxynucleotides in the reactions, Sanger sequencing is also called "dideoxy" sequencing.