Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

126
Chapter 8
How to Obtain a Clone
of a Specific Gene
Chapter contents
CHAPTER CONTENTS
8.1 The problem of selection
8.2 Direct selection
8.3 Identification of a clone from a gene library
8.4 Methods for clone identification
In the preceding chapters we have examined the basic methodology used to clone genes,
and surveyed the range of vector types that are used with bacteria, yeast, plants, and animals.
Now we must look at the methods available for obtaining a clone of an individual,
specified gene. This is the critical test of a gene cloning experiment, success or failure
often depending on whether or not a strategy can be devised by which clones of the
desired gene can be selected directly, or alternatively, distinguished from other recombinants.
Once this problem has been resolved, and a clone has been obtained, the molecular
biologist is able to make use of a wide variety of different techniques that will
extract information about the gene. The most important of these will be described in
Chapters 10 and 11.
8.1 The problem of selection
The problem faced by the molecular biologist wishing to obtain a clone of a single,
specified gene was illustrated in Figure 1.4. Even the simplest organisms, such as E. coli,
contain several thousand genes, and a restriction digest of total cell DNA produces
not only the fragment carrying the desired gene, but also many other fragments carrying
all the other genes (Figure 8.1a). During the ligation reaction there is no selection
for an individual fragment: numerous different recombinant DNA molecules are produced,
all containing different pieces of DNA (Figure 8.1b). Consequently, a variety of
Gene Cloning and DNA Analysis: An Introduction. 6 th edition. By T.A. Brown. Published 2010 by
Blackwell Publishing.