Biomedical Engineering Reference
In-Depth Information
Plate up to 5 10
4
recombinant
phage on 9 cm square Petri dish
Incubate for 6 - 8 h (small plaques),
or overnight (if larger plaques desired)
Cool at 4
°
C for 1 h to stiffen top agar
or top agarose
Nitrocellulose sheet
Overlay plaques with nitrocellulose
sheet for 30 sec to 2 min
Make reference marks for orientation
of sheet with respect to plate
Lift off sheet car
efull
y
Retain plate
Store at 4
°
C
Phage particles and
recombinant phage DNA
from plaques bind to
nitrocellulose
Autoradiographic
images of
positive plaques
(1) Place sheet on filter paper soaked
in alkali to denature DNA
(2) Neutralize on filter paper soaked
in neutral buffer
(3) Bake at 80
°
C
in vacuo
(4) Hybridize with labelled nucleic
acid probe
(5) Wash, autoradiograph or
otherwise detect label
Pick plugs of agar from retained
plate at positions corresponding
to positive plaques
Isolate recombinant phage
In primary screen of densely-plated
phage library single plaques will not
be identifiable; therefore pick area,
dilute, repeat
Fig. 6.13
Benton and Davis' plaque-lift
procedure.
can be denatured, fixed and hybridized. This method
has the advantage that several identical DNA prints
can easily be made from a single-phage plate: this
allows the screening to be performed in duplicate,
and hence with increased reliability, and also allows
a single set of recombinants to be screened with two
or more probes. The Benton and Davis (1977) proced-
ure is probably the most widely applied method of
library screening, successfully applied in thousands
of laboratories to the isolation of recombinant phage
by nucleic acid hybridization (Fig. 6.13). More re-
cently, however, library presentation and screening
have become increasingly automated. Box 6.3 con-
siders the advantages of gridded reference libraries.
In place of RNA probes, DNA or synthetic oligonu-
cleotide probes can be used. A number of alternative
labelling methods are also available that avoid the use
of radioactivity. These methods involve the incorpor-
ation of chemical labels into the probe, such as digoxi-
genin or biotin, which can be detected with a specific
antibody or the ligand streptavidin, respectively.
Probe design
A great advantage of hybridization for library screen-
ing is that it is extremely versatile. Conditions can be
used in which hybridization is very stringent, so that
only sequences identical to the probe are identified.
This is necessary, for example, to identify genomic
clones corresponding to a specific cDNA or to identify
overlapping clones in a chromosome walk (see below).
Alternatively, less stringent conditions can be used
to identify both identical and related sequences.
This is appropriate where a probe from one species
