Figure 10.1 Southern hybridization.
Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A.
Brown.
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.