Nucleic Acid Techniques in Bacterial Systematics

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1 Nucleic Acid Techniques in Bacterial Systematics Edited by Erko Stackebrandt Department of Microbiology University of Queensland St Lucia, Australia and Michael Goodfellow Department of Microbiology University of Newcastle Newcastle upon Tyne, UK A Wiley-Inter science Publication JOHN WILEY & SONS Chichester New York Brisbane Toronto Singapore

2 Contents Contributors Series Preface Preface Introduction E. Stackebrandt and M. Goodfellow xiii xv xvii xix Chapter 1. Isolation and Purification of Nucleic Acids 1 /. L. Johnson A. Introduction 1 B. Growth and cell lysis 1 (i) Growth conditions and culture age 1 (ii) Cell disruption 3 1. Gram-negative bacteria 3 2. Gram-positive bacteria 3 3. Recalcitrant microorganisms 4 C. DNA isolation 5 (i) Marmur protocol 6 (ii) Cetyltrimethylammonium bromide procedure 8 (iii) Hydroxyapatite procedure / 11 (iv) CsCl centrifugation 12 D. RNA isolation 14 (i) Kirby procedure ' 15 (ii) CsCl centrifugation 17 E. Conclusions 17 F. References 18 Chapter 2. DNA Reassociation Experiments 21 /. L. Johnson A. Introduction 21 B. Reassociation and hybridization kinetics 22 (i) Second-order reaction 22 (ii) Pseudo-first-order reaction 25

3 vi Contents C. Reaction variables and conditions (i) Nucleic acids 1. Genomic complexity 2. Fragment length 3. Labeled nucleic acids 4. DNA quantitation (ii) Temperature 1. Reaction rate 2. Reaction specificity (iii) Reaction components 1. Salts 2. Organic molecules D. Assays for DNA reassociation and RNA hybridization (i) Optical measurements (ii) Duplex or hybrid formation with DNA immobilized on membranes 34 (iii) Hydroxyapatite adsorption 37 (iv) S 2 nuclease 38 (v) Use of ribonuclease in hybridization 39 E. Comparisons of DNA reassociation assays 39 F. References 41 Chapter 3. DNA-rRNA Hybridization 45 R. Kilpper-Balz A. Introduction 45 B. Isolation of rrnas, rdnas and cdnas 47 (i) Disruption of cells Chemical and enzymatic lysis Mechanical lysis by French press or glass beads 48 (ii) Separation of ribosomal RNAs ' Sucrose gradient centrifugation Polyacrylamide slab gel electrophoresis 49 (iii) Cloning arid extraction of rdna 52 (iv) Synthesis of cdna by reverse transcription using rrna as template 52 C. Labeling of rrna, rdna and cdna 52 (i) In vivo labeling 52 (ii) In vitro labeling Labeling of rrna Labeling of rdna and cdna 55 D. DNA-rRNA hybridization methods 55 (i) Techniques widely used in phylogenetic studies Membrane filter competition method 55

4 Contents vii 2. Saturation hybridization Determination of r m ( e) values 58 (ii) Other techniques with phylogenetic applications Direct binding on membrane filters followed by correction of hybridization values Membrane filter hybridization using rdna after cloning of rrna genes DNA-rRNA hybridization in solution and collection of the hybrids on diethylaminoethylcellulose filters 64 E. Perspective 64 F. References 66 Chapter 4. DNA Sequencing in Bacterial Systematics 69 W. Ludwig A. Introduction 69 B. Purification of genomic DNA 71 C. Generation, identification and enrichment of DNA fragments 72 (i) Restriction endonuclease digestion of genomic DNA 73 (ii) Agarose gel electrophoresis 73 (iii) Southern transfer 73 (iv) Hybridization Labeling of the probes Hybridization conditions 76 (v) Sizing of DNA restriction fragments 77 (vi) Enrichment of DNA restriction fragments 77 D. Cloning of DNA restriction fragments 78 (i) Ligation of DNA fragments to vector 78 (ii) Transformation 78 (iii) Screening of transformants 79 E. Subcloning / 79 F. DNA sequencing 81 (i) Template preparation 81 (ii) Denaturation of plasmid DNA 82 (iii) Primer design 83 (iv) Primer annealing 84 (v) Primer extension 84 (vi) Termination 85 (vii) Modifications of the method Modifications Use of Klenow fragment of DNA polymerase I Reverse transcriptase 87 (viii) Possible problems and troubleshooting 88 (ix) Gel electrophoresis and autoradiography 88

5 viii Contents 1. Preparation of the gel 2. Running of the gel 3. Autoradiography G. Sequence comparison (i) Interpretation of autoradiographs (ii) Sequence alignment (iii) Comparative analysis H. Concluding remarks I. References Chapter 5. Direct Sequence Analysis of Small RNAs G. Krupp A. Introduction B. Procedures (i) Preparation and isolation of end-labeled RNA 1. Methods for 5'- 32 P-labeling of RNA 2. Method for 3'- 32 P-labeling of RNA 3. Isolation of the labeled RNAs 4. Preliminary analysis of the isolated RNAs 5. Troubleshooting (end-labeling) (ii) Enzymatic sequencing of RNA (iii) Chemical sequencing of RNA (iv) Gel electrophoresis (v) Interpretation of sequencing gels 1. Enzymatic sequencing 2. Chemical sequencing 3. Troubleshooting C. Materials (i) General (ii) Equipment and chemicals (iii) Enzymes (iv) Radioactive materials D. References Chapter 6. 16S/23S rrna Sequencing D. /. Lane A. Introduction B. Reverse transcriptase sequencing of rrna An abbreviated tour through the recent history of the method C. Reverse transcriptase sequencing General description D. Cell growth and preparation E. RNA preparation (i) Method 1

6 Contents (ii) Method F. Reverse transcription sequencing 126 G. Sequencing notes 130 H. Sequencing primers 132 (i) 16S rrna sequencing primers 134 (ii) 23S rrna sequencing primers 136 (iii) Notes on primer design 138 I. Sequencing gels 140 J. Some final thoughts 142 (i) Data analysis 142 (ii) Polymerase chain reaction 143 (iii) The data crisis 143 K. References 144 Appendices 148 Chapter 7. The Polymerase Chain Reaction 177 S. Giovannoni A. Introduction 177 B. The polymerase chain reaction mechanism 177 (i) An outline of the mechanism 177 (ii) Reaction kinetics 178 (iii) Thermus aquaticus DNA polymerase 180 (iv) The design of amplification primers 181 C. Applications of PCR in microbial phylogeny and systematics 183 (i) Molecular phylogeny of cultivated microorganisms 183 (ii) Analysis of natural microbial populations 184 (iii) Symbiosis research 185 D. Methods 188 (i) DNA preparation 188 (ii) Equipment / 189 (iii) Reaction conditions 190 (iv) Controlling reaction specificity 191 (v) Cloning amplified genes 'Forced'cloning techniques Blunt-end cloning techniques Blunt-end ligation procedure 194 (vi) Direct sequencing of amplified genes 195 (vii) Restriction analysis of amplified genes 196 (viii) PCR as a quantitative assay 197 E. Sources of error using PCR 199 (i) Taq polymerase reading errors 199 (ii) Chimeric gene products 199 (iii) Contamination 200 F. References 201

7 Chapter 8. Development and Application of Nucleic Acid Probes D. A. Stahl and R. Amann A. Introduction B. General considerations for nucleic acid hybridization (i) Melting point (ii) T m versus T d versus T w (iii) Estimation of T m 1. DNA probes of greater than 50 nucleotides (iv) Oligonucleotide probes (v) Additional factors influencing oligonucleotide duplex stability 1. Position of mismatch 2. Mismatch composition 3. Degenerate probe positions 4. General considerations 5. RNA-DNA versus DNA-DNA duplexes C. Probe design (i) Empirical probe design (ii) Rational or directed probe design D. Labeling techniques (i) Methods for labeling probes with radioisotopes 1. Nick translation 2. Random primer labeling 3. Riboprobes 4. Polymerase chain reaction 5. 5'-End labeling with 32 P 6. 3'-End labeling with terminal transferase 7. Purification of labeled products E. Immobilization of nucleic acid on membrane supports (i) RFLP analysis (ii) The 16S rrna and a comparative framework for determinative hybridization (iii) The design of phylogenetically based nucleic acid hybridization probes F. Use of fluorescent probes for determinative microscopy (i) Synthesis of fluorescent oligonucleotides. (ii) Purification of fluorescent oligonucleotides (iii) Cell fixation and preparation of contact slides 1. Fixation 2. Pretreatment of microscope slides 3. Application of cells to the microscope slide 4. Whole-cell hybridization G. References Contents

8 Contents xi Chapter 9. DNA Fingerprinting 249 F. GrimontandP. A. D. Grimont A. Introduction 249 B. Isolation of genomic DNA for restriction analysis 250 (i) Extraction of DNA from Gram-negative bacteria 251 (ii) Extraction of DNA from Gram-positive bacteria Method with lysozyme and proteinase K Method with mutanolysine, pronase, sarcosyl and guanidine isothiocyanate 253 (iii) Purification of DNA 253 (iv) Quality control of DNA Spectrophotometric assay Electrophoretic analysis 255 C. Cleavage of DNA with restriction endonucleases 255 D. Agarose gel electrophoresis of DNA restriction fragments 258 E. Transfer of DNA fragments from gel to membrane (vacuum blotting) 261 F. Preparation of nucleic acid probes for fingerprinting 263 (i) Random cloning of chromosomal DNA fragments 263 (ii) Radioactive labeling of probes ' End-labeling of rrna with 32 P by the exchange reaction Labeling of double-stranded DNA by nick translation 266 G. Hybridization of transferred fragments with probes 268 H. Determination of DNA fragment sizes 272 I. Restriction patterns in molecular taxonomy and epidemiology 274 (i) Restriction profiles of total DNA 274 (ii) rrna gene restriction patterns 274 (iii) Restriction patterns involving other genes 275 J. References 276 Chapter 10. From Macromolecules to Trees 281 D. Penny A. Introduction 281 B. Data and terminology 282 C. Methods for reconstructing trees 288 (i) Parsimony criterion of optimality 289 (ii) The Fitch algorithm for a single column 291 (iii) Branch and bound methods 295 (iv) Quartet methods using original sequences 299 D. Hadamard transformation method 302 (i) Part A: Model to data 303

9 xii Contents (ii) Part B: Partition frequencies back to tree 310 (iii) Part C: Fast method for Hadamard multiplication 314 E. Tree comparison metrics 321 F. Conclusion 323 G. References 323 Index 325

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