Quarterly Reviews of Biophysics

Review Article

A review of DNA sequencing techniques

Lilian T. C.  França  a1, Emanuel  Carrilho  a2 and Tarso B. L.  Kist  a3 c1
a1 Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil and Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil (E-mail: lila@biof.ufrj.br)
a2 Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil (E-mail: emanuel@iqsc.sc.usp.br)
a3 Departamento de Biofísica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, 91501–970, Porto Alegre, RS, Brazil (E-mail: tarso@orion.ufrgs.br)


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.

c1 Author to whom correspondence should be addressed. Tel.: 55 51 3316 7618; Fax: 55 51 3316 7003; E-mail: tarso@orion.ufrgs.br