DNA Sequencing Strategies Automated and

This outstanding lab bench reference to the technology of DNA sequencing offers a collection of concise sequencing strategies and cloning protocols. Concentrates on the most up-to-the-minute automated methods and advanced approaches. Preparing DNA for sequencing, sequencing single- doubled-stranded DNA and their variations, how to optimise the primers used, preparation of DNA sequencing gels and the actual collection of results, labelling of DNA fragments for sequencing and data analysis are among the topics covered.

Wilhelm Ansorge and Hartmut Voss are the authors of DNA Sequencing Strategies: Automated and Advanced Approaches, published by Wiley.

Chapter 1 Sequencing Strategies and Cloning Protocols

1

(54)

Section 1.1 Generation of Random Shotgun Libraries

4

(10)

Unit 1.1.1 Shotgun Cloning of Short (0.8-1.5 kb) DNA Fragments Using Sonication

7

(2)

Unit 1.1.2 Shotgun Cloning of Long (2-8 kb) DNA Fragments by Mechanical Shearing

9

(1)

Unit 1.1.3 Shotgun Cloning of Long (2-8 kb) Fragments by Partial Restriction Digestion

10

(4)

Section 1.2 Directed Cloning and Sequencing Strategies

14

(20)

Unit 1.2.1 Directed Subcloning of Restriction Enzyme Fragment from Cosmid DNA

18

(3)

Unit 1.2.2 Delta Restriction Enzyme Cloning

21

(4)

Unit 1.2.3 Unidirectional Nested Deletions

25

(7)

Unit 1.2.4 Primer Walking

32

(2)

Section 1.3 RANDI, An Efficient Cosmid-Scale DNA Sequencing Strategy

34

(3)

Section 1.4 Transformation of Competent Bacteria by Electroporation

37

(4)

Section 1.5 Rapid Precipitation of DNA

41

(2)

Section 1.6 Selected Plasmid Cloning Vectors for DNA Sequencing

43

(10)

Section 1.7 Selected E. coli Strains as Hosts in Cloning for DNA Sequencing

53

(2)

Chapter 2 Preparation of Templates

55

(50)

Section 2.1 Preparation of Single-Stranded M13-DNA (I)-PEG/NaCI

56

(2)

Section 2.2 Preparation of Single-Stranded M13-DNA (II)-Glass Fiber Filters

58

(3)

Section 2.3 Preparation of Plasmid DNA

61

(15)

Unit 2.3.1 Minipreparation of Plasmid DNA Using Qiagen Tip20

63

(4)

Unit 2.3.2 Midi- and Maxipreparation of Plasmid DNA Using Nucleobond AX Columns

67

(5)

Unit 2.3.4 Automated DNA Plasmid Preparation Using the Autogen 740

72

(4)

Section 2.4 Isolation of Human Genomic DNA for PCR Amplification

76

(6)

Unit 2.4.1 DNA Extraction from Hair Roots Using Chelex 100

76

(2)

Unit 2.4.2 Extraction of Human DNA from Periphal Blood Using the QIAamp Blood Kit

78

(2)

Unit 2.4.3 Genomic DNA Extraction from Human Whole Blood Using the Genomix Blood Kit

80

(2)

Section 2.5 Sequencing of PCR Products

82

(23)

Unit 2.5.1 Generation of Recombinant and Genomic PCR Products

82

(11)

Unit 2.5.2 Solid-Phase Preparation and Sequencing Using Dynabeads M280 Streptavidin

93

(4)

Unit 2.5.3 Manifold Sequencing: Efficient Processing of Large Sets of Sequencing Reactions

97

(4)

Unit 2.5.4 Direct Sequencing of Double-Stranded PCR Products

101

(4)

Chapter 3 DNA Sequencing Protocols

105

(30)

Section 3.1 Sequencing Protocols for T7 DNA Polymerase

105

(6)

Unit 3.1.1 Sequencing Single-Stranded DNA with Fluorescently Labeled Universal Primer

107

(1)

Unit 3.1.2 Sequencing Single-Stranded DNA with Internal Labeling

108

(1)

Unit 3.1.3 Sequencing Double-Stranded DNA with Labeled Primers

109

(1)

Unit 3.1.4 Sequencing Double-Stranded DNA with Internal Labeling

109

(2)

Section 3.2 Cycle Sequencing Protocols with Thermostable DNA Polymerases

111

(4)

Unit 3.2.1 Protocol for Sequencing with Fluorescently Labeled Primer Using ThermoSequenase

112

(3)

Section 3.3 Simultaneous Sequencing on Both Strands of Double-Stranded DNA Using the EMBL 2-Dye DNA Sequencer

115

(6)

Unit 3.3.1 Simultaneous Sequencing on Both Strands of Double-Stranded DNA with Two Differently Labeled Primers Using T7 DNA Polymerase

116

(1)

Unit 3.3.2 Simultaneous Sequencing on Both Strands of Double-Stranded DNA with Internal Labeling by Two Differently Labeled Nucleotides Using T7 DNA Polymerase

117

(1)

Unit 3.3.3 Simultaneous Sequencing on Both Strands of Double-Stranded DNA with Two Differently Labeled Primers Using ThermoSequenase

118

(3)

Section 3.4 Sequencing Control Reactions and Troubleshooting

121

(4)

Section 3.5 Labeling DNA Fragments in Automated DNA Sequencing

125

(1)

Section 3.6 Chemical Degradation on Solid Support

126

(5)

Section 3.7 Automated DNA Sequencing Reactions Using the Biomek 1000/2000-SL

131

(4)

Chapter 4 Selection, Synthesis, and Purification of Primers

135

(28)

Section 4.1 Selection of Sequencing Primers

135

(7)

Section 4.2 List of Standard Sequencing Primers

142

(1)

Section 4.3 Purification and Analysis of Sequencing and PCR Primers

143

(8)

Unit 4.3.1 Cleavage of the Oligonucleotide from the Support, Deprotection of the Functional Groups, and Precipitation

144

(1)

Unit 4.3.2 Desalting of Primers Using NAP-5 Columns

145

(2)

Unit 4.3.3 Analysis and Purification of Primers Using Polyacrylamide Gel-Elektrophoresis (PAGE)

147

(2)

Unit 4.3.4 Analysis and Purification of Primers Using HPLC

149

(2)

Section 4.4 Quality Control of Primers

151

(5)

Unit 4.4.1 Quality Control of Primers on Thin-Layer Chromatography (TLC)

152

(1)

Unit 4.4.2 Quality Control of Primers on HPLC

153

(2)

Unit 4.4.3 Manual Product Detritylation and Extraction after Cleavage from Solid-Phase Support

155

(1)

Section 4.5 Basic Synthesis Chemistry, Principle of the EMBL DNA Synthesizer

156

(7)

Unit 4.5.1 Basic Chemistry

157

(3)

Unit 4.5.2 Basic Principles of the EMBL DNA Synthesizer

160

(3)

Chapter 5 Preparation of DNA Sequencing Gels and Electrophoresis

163

(6)

Chapter 6 Computer Analysis

169

(30)

Section 6.1 Fragment Assembly

169

(13)

Unit 6.1.1 Gelstart

170

(2)

Unit 6.1.2 Gelenter

172

(1)

Unit 6.1.3 Geloverlap

172

(2)

Unit 6.1.4 Gelassemble

174

(2)

Unit 6.1.5 Gelstatistics

176

(5)

Unit 6.1.6 Gel Maintenance

181

(1)

Section 6.2 DNA Sequence Analysis

182

(17)

Unit 6.2.1 Search for Open Reading Frames (ORF)

183

(4)

Unit 6.2.2 Dotplot

187

(2)

Unit 6.2.3 Digest

189

(4)

Unit 6.2.4 Base Composition

193

(6)

Index

199

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DNA Sequencing Strategies Automated and Advanced Approaches

Summary

Author Biography

Table of Contents

Supplemental Materials