DNA Sequencing Strategies Automated and Advanced Approaches,9780471136835

DNA Sequencing Strategies Automated and Advanced Approaches

by ; ; ;
Edition: 1st
ISBN13: 9780471136835
ISBN10: 0471136832
Format: Paperback
Pub. Date: 1996-11-12
Publisher(s): Wiley-Liss
List Price: $150.63

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Summary

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.

Author Biography

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

Table of Contents

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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