Northerns, Westerns, & Cloning:
"Molecular Searching Techniques"
searches use one of several forms of complementarity to identify the macromolecules
of interest among a large number of other molecules. Complementarity is
the sequence-specific or shape-specific molecular recognition that occurs
when two molecules bind together. For example: the two strands of a DNA
double-helix bind because they have complimentary sequences; also, an
antibody binds to a region of a protein molecule because they have complimentary
between a probe molecule and a target molecule can result in the formation
of a probe-target complex. This complex can then be located if the probe
molecules are tagged with radioactivity or an enzyme. The location of
this complex can then be used to get information about the target molecule.
hybrid molecular complexes (usually called hybrids) of the following types
can exist (other combinations are possible):
DNA-DNA. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded,
base-paired hybrid with a ssDNA target if the probe sequence is the reverse
complement of the target sequence.
A single-stranded DNA (ssDNA) probe molecule can form a double-stranded,
base-paired hybrid with an RNA (RNA is usually a single-strand) target
if the probe sequence is the reverse complement of the target sequence.
Protein-Protein. An antibody probe molecule (antibodies are proteins)
can form a complex with a target protein molecule if the antibody's antigen-binding
site can bind to an epitope (small antigenic region) on the target protein.
In this case, the hybrid is called an 'antigen-antibody complex' or 'complex'
two important features of hybridization:
reactions are specific - the probes will only bind to targets with complimentary
sequence (or, in the case of antibodies, sites with the correct 3-d shape).
reactions will occur in the presence of large quantities of molecules
similar but not identical to the target. That is, a probe can find one
molecule of target in a mixture of zillions of related but non-complementary
In Situ Hybridization)
allow you to use hybridization to perform a molecular search for one DNA
molecule, or one RNA molecule, or one protein molecule in a complex mixture
containing many similar molecules.
are necessary because a cell contains tens of thousands of genes, thousands
of different mRNA species, and thousands of different proteins. When the
cell is broken open to extract DNA, RNA, or protein, the result is a complex
mixture of all the cell's DNA, RNA, or protein. It is impossible to study
a specific gene, RNA, or protein in such a mixture with techniques that
cannot discriminate on the basis of sequence or shape. Hybridization techniques
allow you to pick out the molecule of interest from the complex mixture
of cellular components and study it on its own.
named for the target molecule.
DNA cut with restriction enzymes - probed with radioactive DNA.
(Click to view an illustration of Southern
RNA - probed with radioactive DNA or RNA.
Protein - probed with radioactive or enzymatically-tagged antibodies.
of hybrids in solution is of little experimental value - if you mix a
solution of DNA with a solution of radioactive probe, you end up with
just a radioactive solution. You cannot tell the hybrids from the non-hybridized
molecules. For this reason, you must first physically separate the mixture
of molecules to be probed on the basis of some convenient parameter.
must then be immobilized on a solid support, so that they will remain
in position during probing and washing. The probe is then added, the non-specifically
bound probe is removed, and the probe is detected. The place where the
probe is detected corresponds to the location of the immobilized target
In the case
of Southern, Northern, and Western blots, the initial separation of molecules
is done on the basis of molecular weight. (Cloning uses a different technique.)
the process has the following steps, detailed below:
This is a
technique that separates molecules on the basis of their size.
slab of gel material is cast. Gels are usually cast from agarose or poly-acrylamide.
These gels are solid and consist of a matrix of long thin molecules forming
sub-microscopic pores. The size of the pores can be controlled by varying
the chemical composition of the gel. The gel is cast soaked with buffer.
The gel is
then set up for electrophoresis in a tank holding buffer and having electrodes
to apply an electric field.
(Click to view an illustration of Gel
The pH and
other buffer conditions are arranged so that the molecules being separated
carry a net (-) charge so that they will me moved by the electric field
from left to right. As they move through the gel, the larger molecules
will be held up as they try to pass through the pores of the gel, while
the smaller molecules will be impeded less and move faster. This results
in a separation by size, with the larger molecules nearer the well and
the smaller molecules farther away.
this separates on the basis of size, not necessarily molecular weight.
For example, two 1000 nucleotide RNA molecules, one of which is fully
extended as a long chain (A); the other of which can base-pair with itself
to form a hairpin structure (B).
(Click to view an illustration of the process
As they migrate
through the gel, both molecules behave as though they were solid spheres
whose diameter is the same as the length of the rod-like molecule. Both
have the same molecular weight, but because B has secondary (2') structure
that makes it smaller than A, B will migrate faster than A in a gel. To
prevent differences in shape (2' structure) from confusing measurements
of molecular weight, the molecules to be separated must be in a long extend
rod conformation - no 2' structure. In order to remove any such secondary
or tertiary structure, different techniques are employed for preparing
DNA, RNA and protein samples for electrophoresis.
If these conditions
are satisfied, the molecules will be separated by molecular weight, with
the high molecular weight molecules near the wells and the low molecular
weight molecules far from the wells. The distance migrated is roughly proportional
to the log of the inverse of the molecular weight (the log of 1/MW). Gels
are normally depicted as running vertically, with the wells at the top and
the direction of migration downwards. This leaves the large molecules at
the top and the smaller molecules at the bottom. Molecular weights are measured
with different units for DNA, RNA, and protein:
DNA for Southern Blots
- DNA is
first cut with restriction enzymes and the resulting double-stranded
DNA fragments have an extended rod conformation without pre-treatment.
RNA for Northern Blots
RNA is single-stranded, RNA molecules often have small regions that
can form base-paired secondary structures. To prevent this, the RNA
is pre-treated with formaldehyde.
Proteins for Western Blots
have extensive 2' and 3' structures and are not always negatively charged.
Proteins are treated with the detergent SDS (sodium dodecyl sulfate)
which removes 2' and 3' structure and coats the protein with negative
- DNA: Molecular
weight is measured in base-pairs, or bp, and commonly in kilobase-pairs
(1000bp), or kbp.
- RNA: Molecular
weight is measured in nucleotides, or nt, and commonly in kilonucleotides
(1000nt), or knt. [Sometimes, bases, or b and kb are used.]
Molecular weight is measured in Daltons (grams per mole), or Da, and
commonly in kiloDaltons (1000Da), or kDa.
On most gels,
one well is loaded with a mixture of DNA, RNA, or protein molecules of
known molecular weight. These 'molecular weight standards' are used to
calibrate the gel run and the molecular weight of any sample molecule
can be determined by interpolating between the standards. Below is a gel
stained with a dye: a colored molecule which binds to a specific class
of macromolecules in a sequence-independent manner (probes bind in a sequence-dependent
contains only one size class of macromolecule - it could be a plasmid,
a pure mRNA transcript, or a purified protein. In this case, you would
not have to use a probe to detect the molecule of interest since there
is only one type of molecule present. Blotting is usually necessary for
samples that are not complex mixtures. By interpolation, its molecular
weight is roughly 3.
is what a sample of total DNA cut with a restriction enzyme, total cellular
RNA, or total cellular protein would look like in a gel stained with a
sequence-independent stain. There are so many bands that it is impossible
to find the one we are interested in. Without a probe (which acts like
a sequence-dependent stain) we cannot get very much information from a
(Click to view a sample
stains and staining procedures are used for different classes of macromolecules:
- DNA is
stained with ethidium bromide (EtBr), which binds to nucleic aids. The
DNA-EtBr complex fluoresces under UV light.
- RNA is
stained with ethidium bromide (EtBr), which binds to nucleic aids. The
RNA-EtBr complex fluoresces under UV light.
is stained with Coomassie Blue (CB). The protein-CB complex is deep
blue and can be seen with visible light.
to Solid Support
DNA, RNA, or protein has been separated by molecular weight, it must be
transferred to a solid support before hybridization. (Hybridization does
not work well in a gel.) This transfer process is called blotting and
is why these hybridization techniques are called blots. Usually, the solid
support is a sheet of nitrocellulose paper (sometimes called a filter
because the sheets of nitrocellulose were originally used as filter paper),
although other materials are sometimes used. DNA, RNA, and protein stick
well to nitrocellulose in a sequence-independent manner.
RNA, or protein can be transferred to nitrocellulose in one of two ways:
which takes advantage of the molecules' negative charge.
blotting, where the molecules are transferred in a flow of buffer
from wet filter paper to dry filter paper.
Note: In a Southern Blot, the DNA molecules in the gel are double-stranded,
so they must be made single stranded in order for the probe to hybridize
to them. To do this, the DNA is transferred using a strongly alkaline
buffer, which causes the DNA strands to separate - this process is called
denaturation - and bind to the filter as single-stranded molecules. RNA
an protein are run in the gels in a state that allows the probe to bind
without this pre-treatment.
At this point,
the surface of the filter has the separated molecules on it, as well as
many spaces between the lanes, etc., where no molecules have yet bound.
If we added the probe directly to the filter now, the probe would stick
to these blank parts of the filter, like the molecules transferred from
the gel did. This would result in a filter completely covered with probe
which would make it impossible to locate the probe-target hybrids. For
this reason, the filters are soaked in a blocking solution which contains
a high concentration of DNA, RNA, or protein. This coats the filter and
prevents the probe from sticking to the filter itself. During hybridization,
we want the probe to bind only to the target molecule.
DNA probes for Southerns and Northerns
is to create a radioactive copy of a double-stranded DNA fragment. The
process usually begins with a restriction fragment of a plasmid containing
the gene of interest. The plasmid is digested with particular restriction
enzymes and the digest is run on an agarose gel. Since a plasmid is usually
less than 20 kbp long, this results in 2 to 10 DNA fragments of different
lengths. If the restriction map of the plasmid is known, the desired band
can be identified on the gel. The band is then cut out of the gel and
the DNA is extracted from it. Because the bands are well separated by
the gel, the isolated DNA is a pure population of identical double-stranded
view an illustration of Microarray
The DNA restriction
fragment (template) is then labeled by Random Hexamer Labeling.:
1) The template
DNA is denatured - the strands are separated - by boiling.
2) A mixture
of DNA hexamers (6 nucleotides of ssDNA) containing all possible sequences
is added to the denatured template and allowed to base-pair. They pair
at many sites along each strand of DNA.
3) DNA polymerase
is added along with dATP, dGTP, dTTP, and radioactive dCTP. Usually, the
phosphate bonded to the sugar (the a-phosphate, the one that is incorporated
into the DNA strand) is synthesized from phosphorus-32 (32P), which is
4) The mixture
is boiled to separate the strands and is ready for hybridization.
to view an illustration of the process)
a radioactive single-stranded DNA copy of both strands of the template
for use as a probe.
Antibodies for Westerns
are raised by injecting a purified protein into an animal, usually a rabbit
or a mouse. This produces an immune response to that protein. Antibodies
isolated from the serum (blood) of that rabbit will bind to the protein
used for immunization. These antibodies are protein molecules and are
not themselves radioactive.
labeled by chemically modifying the side chains of tyrosines in the antibody
with iodine-125 (125I), which is radioactive. A set of enzymes catalyzes
the following reaction:
+ 125I- + H2O2 ---------> H2O + 125iodo-tyrosine-antibody
Antibodies for Westerns
against a particular protein are raised as above and labeled by chemically
cross-linking the antibody molecules to molecules of an enzyme. The resulting
antibody-enzyme conjugate is still able to bind to the target protein.
In all three
blots, the labeled probe is added to the blocked filter in buffer and
incubated for several hours to allow the probe molecules to find their
have formed between the probe and target, it is necessary to remove any
probe that is on the filter that is not stuck to the target molecules.
Because the nitrocellulose is absorbent, some of the probe soaks into
the filter and must be removed. If it is not removed, the whole filter
will be radioactive and the specific hybrids will be undetectable.
To do this,
the filter is rinsed repeatedly in several changes of buffer to wash off
any un-hybridized probe.
Southerns and Northerns, hybrids can form between molecules with similar
but not necessarily identical sequences (For example, the same gene from
two different species.). This property can be used to study genes from
different organisms or genes that are mutated. The washing conditions
can be varied so that hybrids with differing mismatch frequencies are
maintained. This is called 'controlling the 'stringency' - the higher
the wash temperature, the more stringent the wash, the fewer mismatches
per hybrid are allowed.
the Probe-Target Hybrids
At this point,
you have a sheet of nitrocellulose with spots of probe bound wherever
the probe molecules could form hybrids with their targets. The filter
now looks like a blank sheet of paper - you must now detect where the
probe has bound.
If the probe
is radioactive, the radioactive particles that it emits can expose X-ray
film. If you press the filter up against X-ray film and leave it in the
dark for a few minutes to a few weeks, the film will be exposed wherever
the probe bound to the filter. After development, there will be dark spots
on the film wherever the probe bound.
If an antibody-enzyme
conjugate was used as a probe, this can be detected by soaking the filter
in a solution of a substrate for the enzyme. Usually, the substrate produces
an insoluble colored product (a chromogenic substrate) when acted upon
by the enzyme. This produces a deposit of colored product wherever the
view the summary)
a Gene by Hybridization:
In this case,
'to clone the actin gene from humans' means "to end up with a plasmid
which contains a fragment of human DNA which includes the actin gene".
The usual starting point is a plasmid clone of the actin gene from another
organism and human chromosomal DNA. DNA-DNA hybridization is usually used
Southern blotting involves DNA-DNA hybridization, it is not a useful procedure
for cloning a gene. If we were to cut human DNA with a restriction enzyme
and run it on a Southern blot probed with a clone of the actin gene from
an other organism, we could construct a restriction map of the human actin
gene. However, we can not isolate the human actin gene DNA from either
the gel or the filter because at each molecular weight on the gel, there
are many bands of the same length but different sequences.
reason, separating the DNA fragments by molecular weight is unsuitable.
Instead, we separate them by sequence - by making a library, we end up
with a collection of plasmids, physically separated, each containing a
different fragment of human DNA.
Here is a
typical procedure: cloning the human gene for actin, given a clone of
the yeast actin gene.
genomic (chromosomal) DNA from human cells.
a plasmid library of human DNA restriction fragments. This results in
a collection of bacterial colonies, each containing a different plasmid
with a different inserted piece of human DNA.
view an illustration of plasmid
the colonies on agar plates and let them grow. These are called the master
a piece of nitrocellulose onto each master plate and lift off. This leaves
some of each colony on the plate and a replica on the filter.
open (lyse) the bacteria on the filter under conditions that make their
plasmid DNA single-stranded, and bind the DNA onto the filter. There are
now spots of single-stranded plasmid DNA on the filter. These spots correspond
to the locations of the colonies of bacteria on the master plate.
steps 5 through 8 of the Southern Blot,
using a labeled restriction fragment of the yeast actin gene from the
plasmid as a probe. The result will be a piece of X-ray film with a dark
spot corresponding to a place on the filter where DNA from the human actin
gene was present. This spot on the filter corresponds to a colony from
the master plate.
up some of the bacteria from the appropriate colony, grow them in broth
and extract their plasmid DNA. It contains a fragment of human DNA containing
the actin gene.
It is also
possible to clone gene X using an antibody against the protein produced
by gene X. In this case, you make an expression library - a library where
the vector contains a strong E. coli promoter and an ATG codon. Cells
containing these plasmids will produce large amounts of protein from whichever
human gene they carry. If you prepare the filter replicas of the library
as above, you can probe them with an antibody to find the colony that
contains a plasmid that expresses protein X. This plasmid will contain
2001 by Team C0123260
The Legenders , RJC, Singapore