Genotyping of single nucleotide substitutions

The description of the polymerase chain reaction in 1985 caused a revolution in genetics and today molecular diagnostics is one of the leading growth areas across all disciplines of laboratory medicine. This paper reviews the principles and limitations of a number of traditional and emerging techniques for typing of single nucleotide substitutions. The techniques discussed include traditional approaches such as restriction enzyme analysis, more recent homogenous methods, such as those utilising TaqMan, fluorescence resonance energy transfer (FRET) and Scorpion probes, and high resolution melting curve analysis. Non-homogenous but highly flexible approaches such as Pyrosequencing and mass-spectrometry are also discussed. While many techniques are available, it is clear that no one approach is clearly superior. However, in terms of their many advantages and continuing developments, homogenous approaches have much to recommend them.