Biology Reference
In-Depth Information
Once a probe is obtained and labeled, it is used in DNA hybridization experi-
ments to identify those clones that contain the DNA of interest.
Hybridization
involves immobilizing DNA samples from different clones on a solid support
(such as a nitrocellulose or nylon membrane) and then probing the unknown
DNA with a labeled DNA or RNA sequence to identify the clones that contain
the sequence of interest. Identification is possible because the labeled probe
can base-pair with the desired DNA and then be detected by autoradiography
or other methods. There are several different DNA hybridization techniques,
including the Southern-blot analysis described in Chapter 5. Another DNA
hybridization technique is plaque screening.
Figure 6.10
illustrates
plaque screening
of
E. coli
. First,
E. coli
cells are infected
with
λ
that contain exogenous DNA and allowed to grow on an agar substrate.
A nitrocellulose filter is laid onto the
E. coli
lawn and plaques. The precise orien-
tation of the filter is marked. Some of the phage in the plaques containing the
phage are adsorbed onto the filter, where they release their DNA. The DNA is
Figure 6.10
Plaque screening can locate specific genes. Plaque screening involves
in situ
hybridiza-
tion of
E. coli
that have been transformed with a
λ
vector.
Escherichia coli
infected with recombinant
phage are plated out. A nitrocellulose filter is laid on top of the bacterial lawn and plaques. Some
of the phage in a plaque adsorb to the filter. The filter is treated with an alkali to denature the
phage DNA, neutralized, baked in an oven to immobilize the DNA, and placed in a solution with a
radiolabeled DNA probe. The probe base pairs with sequences in the DNA that are complementary
and identifies plaques that may contain the gene of interest. It is then possible to go back to the
plate, pick a few phage from those plaques, and multiply them in
E. coli
.
