Molecular epidemiology : a practical approach /

List of protocols p. xiii
Abbreviations p. xv
Introduction to molecular epidemiology Charles S. Rabkin and Nathaniel Rothman p. xvii
References p. xix
1 Epidemiological methods for studies of genetic factors that influence infectious diseases Eric A. Engels and Thomas R. O'Brien p. 1
1 General considerations p. 1
2 Types of epidemiological studies p. 2
3 Cohort study p. 4
Cohort studies: cumulative incidence p. 5
Cohort studies: incidence p. 7
Example of cohort study p. 8
Attributable risk p. 9
4 Case-control studies p. 10
Example of case-control study p. 12
5 Cross-sectional studies p. 14
6 Interpretation of epidemiological associations p. 15
7 Confouning p. 17
8 Bradford Hill's criteria for causality p. 18
9 Statistical considerations p. 20
10 Further statistical considerations p. 21
11 Interaction p. 23
12 Identifying candidate genes for epidemiological studies p. 24
13 The future of genetic epidemiology p. 25
Reference p. 26
2 Molecular differentiation of bacterial strains Eric W. Brown p. 29
1 Introduction p. 29
2 PCR-based fingerprinting of the bacterial chromosome p. 33
Preparation of genomic DNA from Gram negative and Gram positive eubacterial species p. 34
Reaction conditions p. 37
The 'ribotype' method p. 38
Repetitive PCR fingerprinting p. 40
RAPD-PCR p. 41
Gel electrophoresis of amplicons p. 43
Analysis and interpretation of PCR fingerprint data p. 44
3 RFLP analysis of the bacterial chromosome p. 44
General considerations p. 46
Preparation of genomic DNA for RFLP analysis p. 46
Enzymes p. 47
Polyacrylamide gel electrophoresis (PAGE) p. 48
Analysis and interpretation of genomic restriction data p. 50
4 PFGE analysis of the bacterial chromosome p. 50
General considerations p. 52
Genomic DNA quality p. 53
Restriction enzymes employed in PFGE p. 54
Electrophoretic conditions p. 55
Analysis and interpretations p. 56
5 DNA sequence analysis of the bacterial ompA gene p. 57
Amplification of the bacterial ompA locus p. 58
Cloning and DNA sequencing of ompA PCR products p. 59
Interpretation of ompA nucleotide sequence variation p. 62
6 Conclusion p. 63
Acknowledgements p. 64
References p. 64
3 Detection and quantification of heterogeneous viral targets: HIV as a model Shirley Kwok p. 67
1 Introduction p. 67
2 Impact of sequence heterogeneity on PCR p. 68
3 Primer and probe design for HIV p. 69
Sequence selection p. 69
Reaction conditions and cycling parameters p. 70
4 Assay sensitivity and specificity p. 70
5 Preventing false positives from PCR carryover p. 71
6 Amplification target: DNA vs. RNA p. 72
7 Qualitative vs. quantitative assays p. 73
8 Importance of internal controls and quantitation standards p. 73
Construction of the IC/QS p. 74
Quantitation of the QS p. 74
9 Evaluation of assay performance p. 75
10 Qualitative HIV DNA assay p. 75
Sample collection for HIV DNA amplification p. 75
DNA extraction from cells p. 77
Preparation of DNA from dried blood spots p. 78
DNA amplification p. 79
Colorimetric microwell plate detection p. 81
Interpretation of results p. 83
11 Quantitative HIV RNA assay p. 83
Sample collection for plasma HIV RNA amplification p. 83
Extraction of viral RNA with GuSCN p. 84
RNA amplification p. 87
Quantification of amplified products p. 88
Interpretation of results p. 90
Controls p. 91
12 Quantitative DNA asay p. 91
Quantification of total genomic DNA p. 91
Quantification of HIV DNA p. 93
13 Real time detection formats p. 93
TaqMan technology p. 93
Dye intercalation p. 94
Acknowledgements p. 95
References p. 95
4 Mutation detection by single-stranded conformation polymorphism and denaturing high performance liquid chromatography Michael Dean and Bernard Gerrard and Rando Allikmets p. 97
1 Introduction p. 97
2 Single-stranded conformation polymorphism (SSCP) and heteroduplex analysis (HA) p. 98
Optimization of the PCR reaction p. 98
SSCP sample preparation p. 100
Optimization of SSCP/HA detection p. 101
Multiplexing p. 101
Interpretation of results p. 102
Applications p. 103
Other methods p. 103
3 Denaturing high performance liquid chromatography (DHPLC) p. 103
Application of DHPLC to mutation detection p. 108
Sensitivity of detection p. 108
Conclusions p. 108
Interpretation of results p. 110
Genotyping p. 110
References p. 110
5 DNA pooling methods for association mapping of complex disease loci Lisa F. Barcellos and Soren Germer and William Klitz p. 113
1 Introduction p. 113
2 Quantification of DNA samples for pooled amplification p. 115
3 Genetic analysis of microsatellite markers using pooled DNA samples p. 117
4 Analysis of pooled microsatellite data p. 121
Correction methods for stutter and differential amplification p. 122
Example of a case-control study using pooled DNA amplification p. 126
DNA pooling using nuclear family-based samples p. 127
Analysis of microsatellite allele image patterns from DNA pools p. 127
5 SNP allele frequency determination using kinetic PCR p. 129
6 Experimental design considerations p. 134
Selection of markers for full genome association screen p. 137
Study guidelines p. 138
Technical considerations p. 139
7 DNA pooling in the era of the human genome sequence p. 140
Acknowledgements p. 141
References p. 141
6 Single-sperm typing: a rapid alternative to family-based linkage analysis Michael Cullen and Mary Carrington p. 145
1 Introduction p. 145
2 Isolation of single sperm p. 147
3 Whole genome amplification of haploid DNA p. 152
4 PCR amplification using PEP reaction products as a source of DNA p. 154
Amplification of polymorphic short tandem repeats (STRs) p. 155
Amplification of single nucleotide polymorphisms (SNPs) p. 162
5 Discriminating alleles by gel electrophoresis p. 167
6 Interpretation of sperm typing data p. 172
Single-sperm haplotype scoring p. 173
General approaches for analysing recombination data p. 174
References p. 177
7 PCR-based methods of HLA typing Henry A. Erlich and Elizabeth A. Trachtenberg p. 181
1 Introduction p. 181
2 The HLA loci p. 182
3 HLA allelic sequence diversity p. 183
4 Nomenclature p. 184
5 HLA typing: a brief history p. 185
Serological and cellular methods p. 185
DNA-based techniques p. 185
6 HLA typing requirements p. 204
7 The problem of new alleles and of ambiguity p. 204
Acknowledgements p. 205
References p. 205
8 Evolutionary analysis of molecular sequence data Austin L. Hughes and Jack da Silva and Federica Verra p. 209
1 Introduction p. 209
2 Comparing sequences p. 210
Rationale for statistical models p. 210
Comparing amino acid sequences p. 212
Comparing DNA sequences p. 212
Synonymous and non-synonymous substitution p. 213
Mean nucleotide diversity p. 216
3 Reconstructing phylogenies p. 216
Overview of the problem p. 216
Methods of phylogenetic reconstruction p. 219
Testing phylogenies p. 221
4 Applications p. 222
Phylogenetic analysis p. 222
Testing for positive selection p. 224
Nucleotide diversity and population structure p. 228
References p. 231
List of suppliers p. 233
Index p. 239