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E-grāmata: DNA Sequencing

Luke Alphey

Formāts: 224 pages
Izdošanas datums: 14-Jun-2023
Izdevniecība: Bios Scientific Publishers Ltd
ISBN-13: 9781000944266

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Formāts: 224 pages
Izdošanas datums: 14-Jun-2023
Izdevniecība: Bios Scientific Publishers Ltd
ISBN-13: 9781000944266

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The technique of DNA Sequencing lies at the heart of modern molecular biology. Since current methods were first introduced, sequence databases have grown exponentially, and are now an indispensable research tool. This up-to-date, practical guide is unique in covering all aspects of the methodology of DNA sequencing, as well as sequence analysis. It describes the basic methods (both manual and automated) and the more advanced techniques (for example, those based on PCR) before moving on to key applications. The final section focuses on the analysis of sequence data; it details the software available, and explains how the Internet can be used for accessing software and major databases.

By explaining the options available and their merits, DNA Sequencing allows newcomers to the field to decide which method is the most suitable for their application. For experienced sequencers the book is a useful reference source for details of the less common techniques and as a means of updating knowledge.

Abbreviations

Preface

xiii

PART 1: BASIC PRINCIPLES AND METHODS

1. What is DNA Sequencing?

(10)

An introduction

(1)

Nucleic acid structure

(3)

DNA sequencing

(4)

References

(2)

2. Chemical Degradation (Maxam and Gilbert) Method

(4)

A description of the method

(2)

References

(2)

3. Chain Termination (Sanger Dideoxy) Method

(12)

Introduction

(4)

Cycle sequencing

(6)

References

(2)

4. Instrumentation and Reagents

(14)

Getting started - sequencing kits

(1)

Oligonucleotide primers

(3)

Primer design

(2)

Primer design for cycle sequencing

(1)

DNA polymerase

(1)

Label

(3)

dNTPs and ddNTPs

(2)

dITP and 7-deaza-dGTP

(1)

Pyrophosphatase

(1)

References

(2)

5. Template Preparation

(12)

Introduction

(1)

Preparing single-stranded DNA templates

(5)

Preparing double-stranded DNA templates (plasmids)

(1)

PCR products

(3)

Single-stranded DNA templates from PCR products

(2)

Large templates (lambda, cosmids, P1)

(1)

Templates for semi-automated sequencing

(1)

References

(3)

6. Gel Electrophoresis

(10)

Introduction

(1)

Overview

(1)

Reading a sequence autoradiogram

(1)

Gel systems

(2)

Safety

(1)

Gel plates

(1)

Combs

(1)

Width

(1)

Thickness

(1)

Length

(1)

Temperature control

(1)

Reagents

(4)

Long Ranger™

(3)

Glycerol-tolerant gels

(1)

Formamide gels

(1)

Capillary electrophoresis

(1)

References

(2)

7. Nonradioactive Methods

(18)

Introduction

(1)

Semi-automated sequencers

(1)

ABI 377

(6)

Dye terminator chemistry

(2)

Dye primer chemistry

(1)

Optimizing sequencing on the ABI 377

(4)

Template quality

(1)

Primer quality

(1)

Template and primer concentrations

(1)

Removing unincorporated label

(1)

Future developments

(2)

Brighter dyes

(1)

Better electrophoretic resolution

(1)

Better software

(1)

Uniform peak heights

(1)

Increased throughput

(1)

LI-COR

(1)

References

(3)

8 Troubleshooting

(8)

Introduction

(3)

Co-termination

(2)

Secondary structure

(1)

Dirty template

(1)

Sequencing near to the primer

(1)

Incorrect dNTP incorporation

(1)

Reaction conditions

(1)

dITP

(1)

Compressions

(2)

Base analogs

(1)

Formamide gels

(1)

Reference

(1)

PART 2: APPLICATIONS

9. Confirmatory Sequencing

(8)

Introduction

(1)

Checking constructs

(1)

Sequencing allelic variants

(1)

Alternatives to DNA sequencing

(5)

Using restriction endonucleases

(1)

Using oligonucleotide hybridization

(1)

Using PCR

(3)

References

(1)

10. Sequencing PCR Products

(14)

Introduction

(2)

Sequence information from PCR products

(2)

Sequence analysis of PCR products

(1)

Fidelity of other polymerases

100

(1)

Mutant detection by sequencing PCR products

101

(7)

Tailed primers

103

(1)

Custom dye primers

103

(2)

Dye terminators

105

(2)

Confirming the presence of heterozygotes

107

(1)

Sequencing methylated DNA

108

(2)

References

110

(1)

11. Strategies for New Sequence Determination

111

(14)

Introduction

111

(1)

Directed versus nondirected strategies

112

(1)

Primer walking

113

(1)

Restriction endonuclease digestion and subcloning

114

(2)

'Shotgun' methods

116

(1)

Frequently cutting restriction endonucleases

117

(1)

Sonication

117

(1)

DNase I digestion

117

(1)

Transposon-facilitated sequencing

117

(1)

Deletion series

118

(5)

Exonuclease digests too fast or too slow

120

(1)

DNA is completely degraded by exonuclease

121

(1)

Difficulty in cloning deletion products

121

(1)

Deletions using γδ transposon

122

(1)

References

123

(2)

PART 3: SEQUENCE ANALYSIS

12. Introduction to Bioinformatics and the Internet

A. Brass

125

(8)

Introduction

125

(1)

Bioinformatics is a knowledge-based theoretical discipline

125

(1)

Access to bioinformatics tools

126

(1)

Getting access to tools on the Web

126

(1)

Navigating the Web - or how do I find what I want?

127

(2)

Using Web-based tools

129

(1)

E-mail servers

130

(1)

Accessing remote computers to get useful software - anonymous ftp

130

(2)

Good and bad practice

132

(1)

13. Sequence Databases

A. Brass

133

(12)

Background

133

(1)

Primary databases

134

(1)

DNA databases

134

(1)

Genome databases

135

(1)

Protein sequence databases

135

(2)

Protein structure databases

137

(1)

Primary sequence database annotation

138

(3)

Information retrieval systems

141

(1)

Submitting a sequence to a database

142

(3)

14. Sequence Alignment and Database Searches

A. Brass

145

(14)

Introduction

145

(1)

Scoring matrices

145

(2)

Gap penalties

147

(1)

Pairwise sequence alignments

148

(1)

Multiple sequence alignments

149

(1)

Comparing sequences against a database

150

(6)

When is a hit significant?

156

(1)

References

156

(3)

15. Sequencing Projects and Contig Analysis

A. Brass

159

(8)

Introduction

159

(1)

Analyzing clones

159

(1)

Removing the sequence vector

160

(1)

Removing other cloning sequence artifacts

160

(1)

Contig assembly

161

(1)

Predicting protein-coding regions

162

(1)

Coding regions in cDNA

162

(1)

Coding regions in genomic DNA

163

(1)

DNA analysis

164

(1)

Restriction enzyme maps

164

(1)

Promoters and other DNA control sites

165

(1)

RNA secondary structure prediction

166

(1)

References

166

(1)

16. Protein Function Prediction

A. Brass

167

(12)

Introduction

167

(1)

Comparing a protein sequence against a sequence database to determine function

167

(3)

Hydrophobicity, transmembrane helices, leader sequences and sorting

170

(1)

Calculating hydrophobicity profiles

170

(1)

Predicting transmembrane helices

170

(2)

Leader sequences and protein localization

172

(1)

Coiled-coils

172

(1)

Comparing a protein sequence against motif and profile databases to determine function

173

(1)

Motif databases - PROSITE

174

(1)

Profile databases

175

(1)

References

176

(3)

17. Protein Structure Prediction

A. Brass

179

(10)

Introduction

179

(2)

Protein structure resources

181

(1)

Secondary structure prediction

182

(1)

Tertiary structure prediction

183

(1)

Comparison against sequences of known structure

183

(1)

Homology modeling

184

(1)

Threading algorithms and fold recognition

184

(2)

Critical assessment of structure prediction (CASP)

186

(1)

References

187

(2)

Appendices

189

(14)

Appendix A: Glossary

189

(6)

Appendix B: Amino acid and nucleotide codes

195

(2)

Appendix C: Suppliers

197

(6)

Index

203

Dr Luke Alphey

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Permanent link: https://www.kriso.lv/db/97810009442662e.html

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E-grāmata: DNA Sequencing

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