Gene Cloning

Screening DNA Libraries 129
(c)
Hapten label
Specific antibody
conjugate
Probe
Fluorescent labels
Fluorescent dyes or fluorophores now dominate as labels in a range of
applications including DNA sequencing, forensic analysis and DNA-based
diagnostic tests. Fluorophores are coupled to DNA molecules and can be
detected by measuring the fluorescence produced when they are excited
by light. An additional advantage of fluorescent dyes is that they are available
in a range of different colors and so can be combined to detect several
different DNA molecules in a single reaction.
(a)
(c)
(d)
(b)
Figure 5.6 Screening a DNA library by hybridization with a DNA
probe. a) A nylon filter disk is placed onto the surface of an agar plate on which
the library has been plated. Marks are made on the agar plate and the nylon disk
so that they can be realigned later. b) When the nylon filter is lifted off again,
part of the colonies will adhere to the filter in a mirror image copy of the
original plate, which is retained as the master plate. The filter can be placed,
colony side up, on a fresh agar plate and grown overnight if desired. c) The
colonies are then lysed to release the DNA, treated with alkali to separate the
strands and then fixed to the nylon filter (Figure 5.7). The filter can be probed
with a labeled DNA probe which can be detected by autoradiography. d) The
autoradiograph is aligned with the master plate and the colonies containing library
members with a cloned gene complementary to the probe are identified.