Biomedical Engineering Reference
In-Depth Information
Replica plate onto
nitrocellulose disc
placed on agar in
Petri plate
Incubate
Transformant colonies growing
on agar surface
Retain master
plate
Nitrocellulose disc
removed
Reference
set of
colonies
Pick
positive
colony
(1) Lyse bacteria with 0.5N NaOH
(2) Neutralize
(3) Proteinase
(4) Wash
(5) Bake at 80
°
C
(1) Hybridize with
32
P-labelled probe
(2) Autoradiography
Fig. 6.12
Grunstein-Hogness method for
detection of recombinant clones by colony
hybridization.
DNA print
Autoradiograph
it can be applied to very large numbers of clones and,
in the case of cDNA libraries, can be used to identify
clones that are not full-length (and therefore cannot
be expressed).
Grunstein and Hogness (1975) developed a
screening procedure to detect DNA sequences in
transformed colonies by hybridization
in situ
with
radioactive RNA probes. Their procedure can rapidly
determine which colony among thousands contains
the target sequence. A modification of the method
allows screening of colonies plated at a very high
density (Hanahan & Meselson 1980). The colonies
to be screened are first replica-plated on to a nitrocel-
lulose filter disc that has been placed on the surface
of an agar plate prior to inoculation (Fig. 6.12). A
reference set of these colonies on the master plate is
retained. The filter bearing the colonies is removed
and treated with alkali so that the bacterial colonies
are lysed and the DNA they contain is denatured.
The filter is then treated with proteinase K to remove
protein and leave denatured DNA bound to the
nitrocellulose, for which it has a high affinity, in
the form of a 'DNA print' of the colonies. The DNA
is fixed firmly by baking the filter at 80°C. The
defining, labelled RNA is hybridized to this DNA
and the result of this hybridization is monitored by
autoradiography. A colony whose DNA print gives a
positive autoradiographic result can then be picked
from the reference plate.
Variations of this procedure can be applied to
phage plaques ( Jones & Murray 1975, Kramer
et al.
1976). Benton and Davis (1977) devised a method
called
plaque lift
, in which the nitrocellulose filter is
applied to the upper surface of agar plates, making
direct contact between plaques and filter. The plaques
contain phage particles, as well as a considerable
amount of unpackaged recombinant DNA. Both
phage and unpackaged DNA bind to the filter and
