Figure 10.1 Southern hybridization.

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

Figure 10.2 Southern hybridization can be

used to locate the position of a cloned gene

within a recombinant DNA molecule.

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

Figure 10.3 Conventional agarose gel

electrophoresis and its limitations.

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

Figure 10.4 Orthogonal field alternation gel

electrophoresis (OFAGE).

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

Figure 10.5 Chromosomes and in situ

hybridization.

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

Figure 10.6 Chain termination DNA

sequencing.

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

Figure 10.7 Interpreting the autoradiograph

produced by a chain termination sequencing

experiment. Each track contains the

fragments produced by strand synthesis in

the presence of one of the four

dideoxynucleotide triphosphates

(dideoxyNTPs). The sequence is read by

identifying the track that each fragment lies

in, starting with the one that has moved the

furthest, and gradually progressing up

through the autoradiograph.

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

Figure 10.8 The exonuclease activities of

DNA polymerases. (a) The 5¢®3¢ activity has

an important role in DNA repair in the cell, as

it enables the polymerase to replace a

damaged DNA strand. In DNA sequencing this

activity can result in the 5¢ ends of newly-

synthesized strands becoming shortened. (b)

The 3¢®5¢ activity also has an important role

in the cell, as it allows the polymerase to

correct its own mistakes, by reversing and

replacing a nucleotide that has been added in

error (e.g. a T instead of a G). This is called

proofreading. During DNA sequencing, this

activity can result in removal of a

dideoxynucleotide that has just been added

to the newly-synthesized strand, so that chain

termination does not occur.

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

Figure 10.9 Automated DNA sequencing. (a)

For automated sequencing, each

dideoxynucleotide is labelled with a

fluorescent marker. (b) Each

dideoxynucleotide is labelled with a different

fluorochrome, so the chain-terminated

polynucleotides are distinguished as they

pass by the detector. (c) An example of a

sequence printout.

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

Figure 10.10 The basis to thermal cycle

sequencing. A PCR is set up with just one

primer and one of the dideoxynucleotides.

One of the template strands is copied into a

family of chain-terminated polynucleotides.

ddA = dideoxyATP.

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

Figure 10.11 The problem that arises if the

template DNA for chain-termination DNA

sequencing is able to form intrastrand base

pairs. The stem–loop structure that is able to

form blocks synthesis of the new strand.

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

Figure 10.12 One version of DNA

sequencing by the chemical degradation

method.

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

Figure 10.13 Building up a long DNA

sequence from a series of short overlapping

ones.

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

Figure 10.14 Building up a long DNA

sequence by determining the sequences of

overlapping restriction fragments.

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