Discover DNA Sequencing with Sanger Method: Principle, Techniques and Applications

Describe Sanger sequencing

Template is denatured to form single strands, and extended with a polymerase in the presence of dideocynucleorides (ddNTPs) that cause chain termination

Sanger sequencing, also known as chain termination sequencing, is a technique used for DNA sequencing. It was developed by biochemist Frederick Sanger in the 1970s and earned him a Nobel Prize in chemistry in 1980.

The principle behind Sanger sequencing involves using a DNA polymerase to enzymatically extend a primer that has bound to a single-stranded DNA template. The polymerase adds nucleotides to the primer in a sequential manner according to the template strand. The nucleotides added are labeled with fluorescent or radioactive tags.

In addition to the four normal DNA nucleotides, Sanger sequencing also uses modified nucleotides known as chain-terminating dideoxynucleotides (ddNTPs). These ddNTPs lack the 3′-OH group required for the formation of a phosphodiester bond between adjacent nucleotides. Therefore, when a ddNTP is incorporated into a growing DNA chain, it stops further elongation.

Sanger sequencing involves running four separate reactions, each with a different fluorescent or radioactive ddNTP. The products of these reactions are then electrophoresed on a polyacrylamide gel, allowing separation of the DNA fragments based on their size and fluorescent tag.

The result of Sanger sequencing is a chromatogram that displays the sequence of nucleotides in the DNA fragment. The peaks in the chromatogram correspond to each of the four nucleotides found at that position in the DNA sequence. By analyzing the peaks, the sequence of the DNA fragment can be determined.

Overall, Sanger sequencing is a widely used and reliable method of DNA sequencing that has been used to sequence entire genomes, including the human genome.

More Answers: