Molecular Diversity of Environmental Prokaryotes by Thiago Bruce Rodrigues & Amaro Emiliano Trindade Silva

Author:Thiago Bruce Rodrigues & Amaro Emiliano Trindade Silva
Language: eng
Format: epub
Publisher: CRC Press

Section II

Methods

7 Cloning and Expression of Heterologous Genes

Environmental Applications

Márcia Neiva, Edson Júnior do Carmo, and Spartaco Astolfi-Filho

CONTENTS

7.1 Introduction

7.2 Tools for Molecular Cloning

7.2.1 Enzymes

7.2.1.1 Restriction Endonucleases

7.2.1.2 DNA Ligases

7.2.1.3 Polymerases

7.2.1.4 Reverse Transcriptase

7.2.1.5 Alkaline Phosphatase

7.2.1.6 Polynucleotide Kinase

7.2.1.7 Topoisomerases

7.2.1.8 Cre-Recombinases

7.3 Vectors

7.3.1 Molecular Cloning Vectors

7.3.1.1 Plasmid Cloning Vectors

7.3.1.2 Bacteriophage ʎ Derivative as Cloning Vectors

7.3.1.3 Bacteriophage M13 as Vectors

7.3.1.4 Artificial Chromosomes

7.3.2 Expression Vectors

7.3.2.1 Promoter Sequence

7.3.2.2 Ribosome Binding Site

7.3.2.3 The Signal Peptide or Leader Sequence

7.3.2.4 The Fusion Tag Protein Expression

7.3.3 Integrative Vectors

7.4 Host Organisms

7.4.1 Host Manipulations Systems to Enhance Expression

7.4.1.1 The Restriction-Modification System

7.4.1.2 The Recombination System

7.4.1.3 The a-Complementation System

7.4.1.4 Special Hosts for Expression

7.5 Genetic Transformation: Recombinant DNA Insertion into Host Cells

7.6 Basic Methodologies to Genetic Manipulation

7.6.1 Polymerase Chain Reaction

7.6.2 The cDNA Synthesis: Making DNA Using mRNA Molecules

7.6.3 Gene Library Construction

7.6.3.1 Producing DNA Fragments to Be Cloned

7.6.3.2 Ligating Fragments to the Vector

7.7 Library Screening to Specific Genes Isolation and Analysis of Recombinants

7.7.1 Nucleic Acid Hybridization Screening

7.7.2 Direct Screening by Enzyme Function

7.7.3 Immunodetection of a Protein Screening

7.8 Final Considerations

References

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