1. Introduction 111

2. Levinthal's paradox and energy landscapes 115

2.1 Including randomness in the energy function 121

2.2 Some effects of energetic correlations between structurally similar states 126

3. Resolution of problems by funnel theory 128

3.1 Physical origin of free-energy barriers 133

4. Generic mechanisms in folding 138

4.1 Collapse, generic and specific 139

4.2 Helix formation 139

4.3 Nematic ordering 141

4.4 Microphase separation 142

5. Signatures of a funneled energy landscape 145

6. Statistical Hamiltonians and self-averaging 152

7. Conclusions and future prospects 156

8. Acknowledgments 157

9. Appendix: Glossary of terms 157

10. References 158

The current explosion of research in molecular biology was made possible by the profound discovery that hereditary information is stored and passed on in the simple, one-dimensional (1D) sequence of DNA base pairs (Watson & Crick, 1953). The connection between heredity and biological function is made through the transmission of this 1D information, through RNA, to the protein sequence of amino acids. The information contained in this sequence is now known to be sufficient to completely determine a protein's geometrical 3D structure, at least for simpler proteins which are observed to reliably refold when denatured in vitro, i.e. without the aid of any cellular machinery such as chaperones or steric (geometrical) constraints due to the presence of a ribosomal surface (for example Anfinsen, 1973) (see Fig. 1). Folding to a specific structure is typically a prerequisite for a protein to function, and structural and functional probes are both often used in the laboratory to test for the in vitro yield of folded proteins in an experiment.

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.

Max Ferdinand Perutz, molecular biologist; born Vienna 19 May 1914; Director, MRC Unit for Molecular Biology 1947–62; FRS 1954; Reader, Davy Faraday Research Laboratory, Royal Institution 1954–68, Fullerian Professor of Physiology 1973–79; Chairman, MRC Laboratory of Molecular Biology 1962–79; Nobel Prize for Chemistry (jointly) 1962; CBE 1963; Chairman, European Molecular Biology Organisation 1963–69; CH 1975; OM 1988; married 1942 Gisela Peiser (one son, one daughter); died Cambridge 6 February 2002.