1. Summary 1692. Introduction 1703. Sanger's method and other enzymic methods 1703.1 Random approach 1713.2 Direct approach 1713.3 Enzyme technology 1753.4 Sample preparation 1753.5 Labels and DNA labelling 1763.5.1 Radioisotopes 1763.5.2 Chemiluminescent detection 1763.5.3 Fluorescent dyes 1773.6 Fragment separation and analysis 1803.6.1 Electrophoresis 1803.6.2 Mass spectrometry – an alternative 1824. Maxam & Gilbert and other chemical methods 1835. Pyrosequencing – DNA sequencing in real time by the detection of released PPi 1876. Single molecule sequencing with exonuclease 1907. Conclusion 1928. Acknowledgements 1929. References 193The four best known DNA sequencing techniques are reviewed. Important practical issues
covered are read-length, speed, accuracy, throughput, cost, as well as the automation of
sample handling and preparation. The methods reviewed are: (i) the Sanger method and its
most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other
chemical methods; (iii) the PyrosequencingTM method – DNA sequencing in real time by the
detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with
exonuclease (exonuclease digestion of a single molecule composed of a single strand of
fluorescently labelled deoxynucleotides). Each method is briefly described, the current
literature is covered, advantages, disadvantages, and the most suitable applications of each
method are discussed.
1SBP-02 REAL-TIME MONITORING OF BIOMOLECULES IN ZERO-MODE WAVEGUIDES: DNA SEQUENCING AND BEYOND(1SBP Advanced Single Molecule Sequencing System,Symposium,The 51th Annual Meeting of the Biophysical Society of Japan)
