Figure 8.1 The problem of selection.
Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.
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.