Sequencing is a process of determining the precise order of nucleotides in a DNA template after amplification using specific loci. Sanger sequencing was invented by Fredrick Sanger in 1977.It is a widely used sequencing method and now replaced by Next-Generation sequencing methods. In Sanger sequencing, only fragments of DNA (specific locus) can be amplified and sequenced using primers. In Next-Gen sequencing, millions of nucleotides can be sequenced simultaneously.
Sanger sequencing is also known as the chain termination method. In this process, there is selective incorporation of dideoxynucleotide to the sequencing reaction. Dideoxy nucleotide lacks 3′ OH group in the sugar molecule unlike deoxynucleotides that aids in the formation of phosphodiester bond formation between two nucleotides.
image credit – https://www.researchgate.net/figure/The-Sanger-sequencing-method-A-simplified-schematic-of-the-Sanger-sequencing-method_fig8_303565848
Panel a) depicts the comparison of ddNTP and DNTP. In panel b) addition of ddNTP terminates the sequencing reaction. In panel c) DNA fragments are incorporated, sorted by size and sequential nucleotides are determined by last incorporated nucleotide. In panel D) sequences are represented in the form of a chromatogram.
A DNA sample is divided into four separate sequencing reactions. In all sequencing reactions all four standard deoxynucleotides, DNA polymerase was added. The components of sequencing reactions are a) Amplified DNA template, b) Sequencing master mix, c) forward or reverse sequencing primers. In each reaction, one of the dideoxynucleotide triphosphates is added. The amplicons are prepared for sequencing and the sequencing reaction will proceed for 2.5 hours in the thermal cycler according to the manufacturer protocols.
After DNA sequencing, the template is prepared for loading onto a genetic analyzer by adding Hi-Di Formamide. The template is incubated for 5 minutes in a water bath at 95C and cooled for 2 minutes to keep it denatured. The samples are transferred to 96 well plate and capillary electrophoresis takes place in the genetic analyzer.