FISH and DNA sequencing

FISH (fluorescence in situ hybridisation) is used to study chromosomes. A short piece of DNA (a probe) that matches a known sequence is labelled with a fluorescent dye. The chromosomes on a slide are treated to separate their DNA strands (denatured), and the probe sticks (hybridises) wherever its matching sequence is. Under a fluorescence microscope, a glowing spot shows exactly where that sequence lies, revealing missing, extra or moved pieces of chromosomes. In chromosome painting, each chromosome is labelled in its own colour.

DNA sequencing finds the order of bases in DNA. It underpins research, medical diagnosis, forensics, agriculture and the sequencing of whole genomes, including the human genome.

Sanger's chain-termination method, still the gold standard: DNA polymerase extends a primer along the template. Along with normal nucleotides, special dideoxynucleotides (ddNTPs) are added. A ddNTP has no 3'-OH group, so once one is added, the chain must stop. This gives a set of pieces, each ending at a particular base. They are separated by size by PAGE, and the sequence is read from the bottom (smallest) up.

Maxam and Gilbert's method works differently, by chemistry: the DNA is labelled at its 5' end with radioactive ³²P and cut at particular bases with chemicals (dimethyl sulphate, hydrazine), then the pieces are separated by PAGE. It does not build new DNA.

Automated sequencers label each of the four ddNTPs with a different coloured fluorescent dye, so all four reactions can run in one tube, and they use thin capillaries instead of slab gels. A laser reads the colours as they pass, shown as peaks. Next-generation sequencing (platforms such as Roche/454, Illumina and SOLiD) runs millions of reactions in parallel, sequencing far faster and cheaper.