DNA Sequencing (Introduction to Biotechniques)

✍ Scribed by Dr Luke Alphey

• Publisher: Garland Science
• Tongue: English
• Leaves: 221
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Cover
Half Tilte
Series Page
Title Page
Copyright Page
Table of Contents
Preface
Abbreviations
Acknowledgements
Part 1: Basic Principles and Methods
1: What Is DNA Sequencing?
An Introduction
Nucleic Acid Structure
DNA Sequencing
References
2: Chemical Degradation (Maxam and Gilbert) Method
A Description of the Method
References
3: Chain Termination (Sanger Dideoxy) Method
Introduction
Cycle Sequencing
References
4: Instrumentation and Reagents
Getting Started - Sequencing Kits
Oligonucleotide Primers
Primer Design
Primer Design for Cycle Sequencing
DNA Polymerase
Label
dNTPs and ddNTPs
dITP and 7-deaza-dGTP
Pyrophosphatase
References
5: Template Preparation
Introduction
Preparing Single-Stranded DNA Templates
Preparing Double-Stranded DNA Templates (Plasmids)
PCR Products
Single-Stranded DNA Templates from PCR Products
Large Templates (lambda, Cosmids, Pl)
Templates for Semi-Automated Sequencing
References
6: Gel Electrophoresis
Introduction
Overview
Reading a Sequence Autoradiogram
Gel Systems
Safety
Gel Plates
Combs
Width
Thickness
Length
Temperature Control
Reagents
Long Ranger™
Glycerol-Tolerant Gels
Formamide Gels
Capillary Electrophoresis
References
7: Nonradioactive Methods
Introduction
Semi-Automated Sequencers
ABI 377
Dye Terminator Chemistry
Dye Primer Chemistry
Optimizing Sequencing on the ABI 377
Template Quality
Primer Quality
Template and Primer Concentrations
Removing Unincorporated Label
Future Developments
Brighter Dyes
Better Electrophoretic Resolution
Better Software
Uniform Peak Heights
Increased Throughput
LI-COR
References
8: Troubleshooting
Introduction
Co-Termination
Secondary Structure
Dirty Template
Sequencing Near to the Primer
Incorrect DNTP Incorporation
Reaction Conditions
dITP
Compressions
Base Analogs
Formamide Gels
Reference
Part 2: Applications
9: Confirmatory Sequencing
Introduction
Checking Constructs
Sequencing Allelic Variants
Alternatives to DNA Sequencing
Using Restriction Endonucleases
Using Oligonucleotide Hybridization
Using PCR
References
10: Sequencing PCR Products
Introduction
Sequence Information from PCR Products
Sequence Analysis of PCR Products
Fidelity of other Polymerases
Mutant Detection by Sequencing PCR Products
Tailed Primers
Custom Dye Primers
Dye Terminators
Confirming the Presence of Heterozygotes
Sequencing Methylated DNA
References
11: Strategies for New Sequence Determination
Introduction
Directed Versus Nondirected Strategies
Primer Walking
Restriction Endonuclease Digestion and Subcloning
'Shotgun' Methods
Frequently Cutting Restriction Endonucleases
Sonication
DNAse I Digestion
Transposon-Facilitated Sequencing
Deletion Series
Exonuclease Digests Too Fast or Too Slow
DNA Is Completely Degraded by Exonuclease
Difficulty in Cloning Deletion Products
Deletions Using γδ Transposon
References
Part 3: Sequence Analysis
12: Introduction to Bioinformatics and the Internet – A. Brass
Introduction
Bioinformatics Is a Knowledge-based Theoretical Discipline
Access to Bioinformatics Tools
Getting Access to Tools on the Web
Navigating the Web- or How Do I Find What I Want?
Using Web-based Tools
E-mail Servers
Accessing Remote Computers to Get Useful Software – Anonymous Ftp
Good and Bad Practice
13: Sequence Databases – A. Brass
Background
Primary Databases
DNA Databases
Genome Databases
Protein Sequence Databases
Protein Structure Databases
Primary Sequence Database Annotation
Information Retrieval Systems
Submitting a Sequence to a Database
14:. Sequence Alignment and Database Searches - A. Brass
Introduction
Scoring Matrices
Gap Penalties
Pairwise Sequence Alignments
Multiple Sequence Alignments
Comparing Sequences Against a Database
When Is a Hit Significant?
References
15: Sequencing Projects and Contig Analysis – A. Brass
Introduction
Analyzing Clones
Removing the Sequence Vector
Removing Other Cloning Sequence Artifacts
Contig Assembly
Predicting Protein-coding Regions
Coding Regions in cDNA
Coding Regions in Genomic DNA
DNA Analysis
Restriction Enzyme Maps
Promoters and Other DNA Control Sites
RNA Secondary Structure Prediction
References
16: Protein Function Prediction – A. Brass
Introduction
Comparing a Protein Sequence Against a Sequence Database to Determine Function
Hydrophobicity, Transmembrane Helices, Leader Sequences and Sorting
Calculating Hydrophobicity Profiles
Predicting Transmembrane Helices
Leader Sequences and Protein Localization
Coiled-Coils
Comparing a Protein Sequence Against Motif and Profile Databases to Determine Function
Motif Databases - PROSITE
Profile Databases
References
17: Protein Structure Prediction- A. Brass
Introduction
Protein Structure Resources
Secondary Structure Prediction
Tertiary Structure Prediction
Comparison Against Sequences of Known Structure
Homology Modeling
Threading Algorithms and Fold Recognition
Critical Assessment of Structure Prediction (CASP)
References
Appendices
Appendix A: Glossary
Appendix B: Amino Acid and Nucleotide Codes
Appendix C: Suppliers
Index