Biology Reference
In-Depth Information
of
E. coli
, and it has a unique
Eco
RI restriction site near the end of the gene.
A cDNA cloned into this site in the correct orientation and reading frame will
produce a fusion protein. Upon lysis of the
E. coli
cells, the protein is released
and picked up on nitrocellulose in just the same way as in plaque screening. The
plaque containing the interesting cDNA clone can be detected by incubating the
filter with a specific antibody.
It is possible to determine the difference in abundance between two different
mRNA populations. Thus, mRNA produced from different tissues from the same
organism or mRNA produced from a tissue before and after a specific induction
signal can be compared by differential screening. A cDNA library is prepared
from one of the two mRNA populations, and the two copies are immobilized
on filters. The filters are then screened twice, once with highly labeled cDNA
prepared from one of the two mRNA populations and once with a probe from
the other mRNA population. By comparing the signals produced on the two fil-
ters probed with the different probes, it is possible to determine whether mRNA
sequences are present in one population but absent or rare in the alternative
mRNA population. However, this method is used less often now that gene chip
methods are available (see Section 6.9).
6.7 Labeling Probes by a Variety of Methods
Nucleic
-
acid hybridization
is used for many different purposes in molecular
genetics. Formerly, nearly all phases of cloning and characterization or anal-
ysis of DNA involve hybridizing one strand of nucleic acid to another by com-
plementary-base pairing. Nucleic-acid hybridization relies on the fact that two
single-stranded nucleic-acid molecules with complementary bases (DNA with
DNA and DNA with RNA) are able to pair via hydrogen bonds. The length of
the homologous sequences determines the strength of the hybridization, as do
the experimental conditions (temperature and pH) and the degree of sequence
homology. The Southern blot, a nucleic-acid hybridization method, and one
method for labeling probes (nick translation) were described in Chapter 5. Other
nucleic-acid hybridization techniques include colony or plaque hybridization,
and Northern-blot analysis, in which the immobilized nucleic acid is RNA instead
of DNA.
Several labeling methods other than nick translation are available and are
outlined here. The success of nucleic-acid hybridization often relies on methods
to introduce a label into cloned segments of DNA or RNA. Each labeling tech-
nique has optimal sizes, efficiency, and different amounts of nucleic-acid tem-
plate that are required. One measure of the efficiency of radiolabeling is the
