DNA Sequencing

The dideoxy method of DNA sequencing, also known as Sanger sequencing, is a widely used technique developed by Frederick Sanger in 1977. It relies on chain-termination during DNA synthesis. Here's how it works:

Principle of the Dideoxy Method
The method incorporates dideoxynucleotides (ddNTPs) into a growing DNA strand during replication. Unlike normal deoxynucleotides (dNTPs), ddNTPs lack a hydroxyl group (-OH) on the 3' carbon of the sugar. This absence prevents further extension of the DNA strand once a ddNTP is added, effectively terminating the chain.

Steps of the Sanger Sequencing Process
DNA Template Preparation
The DNA to be sequenced is denatured to produce a single-stranded template.

Reaction Setup
The sequencing reaction contains:

A DNA template.
A primer complementary to the 3' end of the template.
DNA polymerase enzyme.
A mix of the four normal deoxynucleotides (dATP, dTTP, dCTP, and dGTP).
A small proportion of each dideoxynucleotide (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a fluorescent dye (modern methods) or a radioactive label (historical methods).
Chain Termination by ddNTPs
During DNA synthesis, DNA polymerase incorporates either a regular dNTP or a ddNTP. If a ddNTP is incorporated, chain elongation stops.

Separation by Size (Electrophoresis)
The resulting DNA fragments of varying lengths are separated by size using polyacrylamide gel electrophoresis (historically) or capillary electrophoresis (modern methods).

Detection and Sequencing

In modern techniques, fluorescently labeled ddNTPs are detected by a laser as they pass through the capillary, and the sequence is read as a series of fluorescence peaks.
The order of the peaks corresponds to the sequence of the DNA template.
Advantages of the Dideoxy Method
High accuracy for sequencing short to medium DNA fragments.
Reliable and easy to interpret.
Was the gold standard for DNA sequencing for decades.
Limitations
Relatively slow and expensive compared to next-generation sequencing (NGS).
Limited to sequencing shorter fragments (typically 500–1000 base pairs per reaction).
Applications
Verifying the sequence of cloned DNA.
Sequencing single genes or small genomes.
Genetic testing and mutation analysis