DNA Sequencing (eBook)

Front Cover 1
DNA Sequencing 4
Copyright Page 5
Contents 8
Acknowledgements 6
Chapter 1. Introduction 14
1.1. Preliminary remarks 14
1.2. Organisation of the book 17
1.3. General background to DNA sequencing 20
Chapter 2. DNA Sequencing 43
2.1. DNA sequencing by primed synthesis methods 43
2.2. Detailed protocol for the 'plus' and 'minus' primed synthesis method 53
2.3. Additional methods useful in conjunction with plus and minus method 59
2.4. Discussion and summary 74
2.5. Sequence analysis of short DNA fragments 77
Chapter 3. Chain terminator sequencing 85
3.1. DNA sequencing with chain terminating inhibitors (Sanger et al., 1977) 85
3.2. Sequencing by partial ribosubstitution 99
3.3. Direct enzymatic method for sequencing double-stranded restriction fragments using dideoxynucleoside triphosphates (Maat and Smith. 1978) 102
3.4. The use of exonuclease Ill for preparing single-stranded DNA for use as a template in the chain terminator sequencing method (Smith, 1979, 1980) 117
Chapter 4. Primed synthesis methods applied to DNA fragments cloned into phage M13 134
4.1. General introduction 134
4.2. Rapid random sequencing by shotgun cloning into single stranded phage vectors (Sanger et al., 1980) 137
4.3. Detailed experimental procedures 174
Experimental procedures 177
4.4. Dideoxy-nucleotide sequencing procedures 200
4.5. Results 203
4.6. Discussion 210
4.7. Hints for reading sequencing gels 213
4.8. Sequencing long single-stranded DNAs cloned in bacteriophage M13 using internal primers 219
4.9. Sequencing cDNA from reverse transcripts of RNA 227
Key references 234
Chapter 5. DNA sequencing by the Maxam-Gilbert chemical procedure 243
5.1. Introduction 243
5.2. End-labelling DNA segments 249
5.3. Separating the two labelled ends of complementary strands 256
5.4. Strand separation 257
5.5. Elution of DNA from polyacrylamide gels 258
5.6. Base-specific chemical cleavage reactions 261
5.7. Gel electrophoresis 265
5.8. Reading the gel 266
5.9. Experimental procedures 268
5.10. Sequencing reactions (Maxam and Gilbert, 1980) 279
5.11. Trouble-shooting guide (Maxam and Gilbert, 1980) 283
5.12. Results 286
5.13. Strategies for sequencing double-stranded DNA 288
Update 299
Update to chapter 4 299
Update to chapter 5 308
Appendices 310
Appendix 1. List of contractions and special terms 310
Appendix 2. Cloning vectors 313
Appendix 3. Commercially obtainable restriction endonucleases (1981) 314
Appendix 4. Autoradiography 315
Appendix 5. Equipment 317
Appendix 6 320
Appendix 7. Nucleotide sequence of M13mp7 7238 residues
Appendix 8. Restriction map of M13mp8 (7229 base pairs) 323
Chapter 6. Computer methods for DNA sequencers 324
1. Introduction 324
2. Data storage and retrieval: the DB system 331
3. Analysis: restriction sites, hairpin loops, translation, homologies 346
4. Choosing hardware and software 359
5. References 360
Appendix to chapter 6 362
Getting started with the DB system 362
Running the programs 363
References 382
Subject index 388