A review of DNA sequencing techniques

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Abstract
1. Summary 169 2. Introduction 170 3. Sanger's method and other enzymic methods 170 3.1 Random approach 171 3.2 Direct approach 171 3.3 Enzyme technology 175 3.4 Sample preparation 175 3.5 Labels and DNA labelling 176 3.5.1 Radioisotopes 176 3.5.2 Chemiluminescent detection 176 3.5.3 Fluorescent dyes 177 3.6 Fragment separation and analysis 180 3.6.1 Electrophoresis 180 3.6.2 Mass spectrometry – an alternative 182 4. Maxam & Gilbert and other chemical methods 183 5. Pyrosequencing – DNA sequencing in real time by the detection of released PPi 187 6. Single molecule sequencing with exonuclease 190 7. Conclusion 192 8. Acknowledgements 192 9. References 193 The 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.

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