Biology Reference
In-Depth Information
CHAPTER 6
Some Additional Tools for the
Molecular Biologist
Chapter Outline
6.1 Overview 215
6.2 Introduction 216
6.3 The Perfect Genomic Library 218
6.3.1 Lambda (
λ
) Phage as a Vector
219
6.3.2 Cloning with Cosmids 226
6.3.3 Cloning in the Filamentous Phage M13
228
6.3.4 Phagemids 230
6.3.5 BACs 230
6.4 cDNA Cloning 230
6.5 Enzymes Used in Molecular Biology Experiments 234
6.6 Isolating a Specific Gene from a Library if Whole-Genome Sequencing is Not Done
234
6.7 Labeling Probes by a Variety of Methods 240
6.7.1 Synthesis of Uniformly Labeled DNA Probes by Random Primers
241
6.7.2 Synthesis of Probes by Primer Extension
241
6.7.3 End-Labeled Probes 241
6.7.4 Single-Stranded Probes
241
6.7.5 Synthetic Probes 242
6.8 Baculovirus Vectors Express Foreign Polypeptides in Insect Cells
242
6.9 Expression Microarray Analysis
243
General References
247
References Cited
247
6.1 Overview
Cloning of DNA has five essential components: 1) a method for generating
exogenous DNA fragments, 2) reactions to join exogenous DNA fragments to
a vector, 3) a method to introduce the vector into a host cell where the vec-
tor ensures the exogenous DNA is replicated, 4) methods for selecting or iden-
tifying the vectors that contain the introduced DNA (recombinant molecules),
and 5) methods for analyzing the cloned DNA. Genomic libraries can be con-
structed in
λ
phage (a bacteriophage that attacks
Escherichia coli
) and other vec-
tors. Complementary DNA (cDNA) libraries can be cloned into various vectors,
