What is a dideoxynucleotide and why is it used in sequencing?

It is a modified nucleotide that, once added, stops further synthesis; incorporating it at random positions generates fragments ending at each base, which reveal the sequence.

How can whole genomes be sequenced so quickly now?

Massively parallel platforms read millions of DNA fragments at once and use software to assemble them, vastly increasing speed and lowering cost compared with earlier methods.

DNA Sequencing

How the order of bases in DNA is determined — from Sanger's chain-termination method to the massively parallel technologies that read whole genomes.

Definition

DNA sequencing is the determination of the precise order of nucleotides in a DNA molecule, achieved historically by chain-terminating synthesis and now predominantly by massively parallel methods that read many fragments simultaneously.

Scope

This topic covers methods for reading the nucleotide sequence of DNA: the chain-termination (Sanger) method and its principle of base-specific termination, and the high-throughput, massively parallel approaches that succeeded it and made genome-scale sequencing routine. It treats the logic of sequencing; amplification and cloning that prepare DNA for sequencing are covered in companion topics.

Core questions

How does chain-termination sequencing reveal the order of bases?
Why did labelled dideoxynucleotides make sequencing practical?
How do massively parallel methods scale sequencing to whole genomes?
How are short reads assembled into longer sequences and genomes?

Key theories

Chain-termination sequencing: Synthesis in the presence of chain-terminating dideoxynucleotides produces a set of fragments ending at each occurrence of a given base, and ordering these by length reveals the sequence, as introduced by Sanger and colleagues.
Massively parallel sequencing: Reading enormous numbers of DNA fragments at once and reconstructing the sequence computationally lowered cost and raised throughput dramatically, enabling routine genome-scale sequencing.

Mechanisms

In chain-termination sequencing, a primer is extended along a template in the presence of normal nucleotides plus a small proportion of dideoxynucleotides that, once incorporated, halt synthesis; the result is a nested set of fragments whose terminal base is known, which are separated by size to read the sequence. Modern massively parallel platforms immobilise and amplify many template fragments and detect base incorporation across all of them simultaneously, generating large numbers of short reads that software aligns or assembles into the complete sequence.

Clinical relevance

Sequencing underlies genetic diagnosis, cancer genomics, pathogen surveillance, and personalised approaches to medicine; offered as significance, not clinical guidance.

History

Sanger's chain-termination method and the parallel Maxam–Gilbert chemical method, both from the 1970s, made DNA sequencing feasible and earned Nobel recognition; the rise of massively parallel technologies in the 2000s reduced costs by orders of magnitude and ushered in the genomic era.

Key figures

Frederick Sanger
Walter Gilbert

Seminal works

sanger1977
lodish2016

Frequently asked questions

What is a dideoxynucleotide and why is it used in sequencing?: It is a modified nucleotide that, once added, stops further synthesis; incorporating it at random positions generates fragments ending at each base, which reveal the sequence.
How can whole genomes be sequenced so quickly now?: Massively parallel platforms read millions of DNA fragments at once and use software to assemble them, vastly increasing speed and lowering cost compared with earlier methods.