The "chain termination method," usually referred to as Sanger sequencing, was created in 1977 by English biochemist Frederick Sanger and his associates.

This technique is intended to identify the nucleotide base sequence in a sample of DNA (commonly less than 1,000 bp in length). The "gold standard" for validating DNA sequences, including those already sequenced using next-generation sequencing, is Sanger sequencing, which has a base accuracy of 99.99 percent (NGS).

A DNA primer complementary to the template DNA (the DNA to be sequenced) is used as the starting point for DNA synthesis during Sanger sequencing. The polymerase extends the primer by adding the complementary dNTP to the template DNA strand when the four deoxynucleotide triphosphates (dNTPs) A, G, C, and T are present. Four dideoxynucleotide triphosphates (ddNTPs)—dATP, ddGTP, ddCTP, and dTTP—each labeled with a different fluorescent dye—are employed to stop the synthesis reaction in order to identify which nucleotide is incorporated into the chain of nucleotides. The ribonucleotide in ddNTPs has had an oxygen atom removed compared to dNTPs, making it incapable of forming a connection with the following nucleotide.

Steps in Sanger Sequencing

Sanger sequencing involves six steps, which are as follows:
(1) Denaturement of the double-stranded DNA (dsDNA) results in the formation of two single-stranded DNA (ssDNA).
(2) Attached to the sequence is a primer that matches one end.
(3) Only one type of ddNTP and four different types of dNTPs are added to each of the four polymerase solutions.
(4) The DNA synthesis reaction starts, and the chain grows until an unpredicted termination nucleotide is added.
(5) Denatured ssDNA is created from the resultant DNA pieces.
(6) Gel electrophoresis is used to separate the denatured fragments and identify their sequencing.

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