Figure 8.1 The problem of selection.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.2 The basic strategies that can be

used to obtain a particular clone: (a) direct

selection; (b) identification of the desired

recombinant from a clone library.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.3 Direct selection for the cloned R6-

5 kanamycin resistance (kanR) gene.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.4 Direct selection for the trpA gene

cloned in a trpA- strain of E. coli.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.5 Preparation of a gene library in a

cosmid vector.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.6 Different genes are expressed in

different types of cell.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.7 One possible scheme for cDNA

cloning (see text for details). Poly(A) =

polyadenosine, oligo(dT) =

oligodeoxythymidine.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.8 Nucleic acid hybridization. (a) An

unstable hybrid molecule formed between

two non-homologous DNA strands. (b) A

stable hybrid formed between two

complementary strands. (c) A DNA–RNA

hybrid, such as may be formed between a

gene and its transcript.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.9 Colony hybridization probing with

a radioactively labelled probe.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.10 Labelling DNA by random

priming. The mixture of random hexamers

(hexameric oligonucleotides of random

sequence) is sufficiently complex to include at

least a few molecules that can base pair with

the probe. dNTP = 2¢-deoxynucleotide 5¢-

triphosphate.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.11 Two methods for the non-

radioactive labelling of DNA probes.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.12 Probing within a library to

identify an abundant clone.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.13 The amino acid sequence of

yeast cytochrome c. The hexapeptide that is

highlighted red is the one used to illustrate

how a nucleotide sequence can be predicted

from an amino acid sequence.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.14 A simplified scheme for

oligonucleotide synthesis. The protecting groups

attached to the 3¢ and 5¢ termini prevent

reactions between individual mononucleotides. By

carefully controlling the times at which the

protecting groups are removed, mononucleotides

can be added one by one to the growing

oligonucleotide.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.15 The use of a synthetic, end-

labelled oligonucleotide to identify a clone of

the yeast cytochrome c gene.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.16 Heterologous probing.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.17 Antibodies. (a) Antibodies in the

bloodstream bind to foreign molecules and

help degrade them. (b) Purified antibodies

can be obtained from a small volume of blood

taken from a rabbit injected with the foreign

protein.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.

Figure 8.18 Using a purified antibody to

detect protein in recombinant colonies.

Instead of labelled protein A, the antibody

itself can be labelled, or alternatively a

second labelled antibody which binds

specifically to the primary antibody can be

used.

Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.