Three generations of DNA sequencing

DNA sequencing reads the order of bases. It has gone through three generations.

First generation. Chromosomes were separated by pulse field gel electrophoresis, cut up, cloned in large vectors (BAC, YAC or PAC), sub-cloned into plasmids, and then sequenced by the chain termination (Sanger) method. The chain-terminated fragments carry fluorescent labels and run through thin capillaries filled with gel; a detector reads each colour as a peak on a chromatogram. Each capillary reads about 800-1000 bases, and a machine with 96 capillaries reads up to 96,000 bases per run. It is accurate, but slow, labour-intensive and costly.

Next (second) generation. Instead of one fragment per capillary, millions are read at once (massively parallel), with no cloning needed. In the Illumina method:

• DNA fragments with adapters attached are bound to a glass flow cell.
• Each fragment is copied in place by bridge amplification, forming a cluster of identical copies.
• Fluorescent nucleotides are added one at a time, and a camera photographs which colour each cluster takes up.

It reads millions of fragments together, but each read is short, only 75-300 bases.

Third generation: nanopores. A protein helicase unwinds the DNA and pushes one strand through a tiny pore (made from a porin-like protein) in a synthetic membrane, while an electric current flows through it. Each base disturbs the ionic current in its own way, so the sequence is read directly, base by base.

Nanopore sequencing is fast and simple, shows results in real time, works with a small device in the field, and reads very long stretches (1 Mb or more), at low cost.