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Southerns, Northerns, Westerns, & Cloning:
"Molecular Searching Techniques"

Complementarity and Hybridization

Molecular 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 shapes.

Complementarity 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.

In solution, hybrid molecular complexes (usually called hybrids) of the following types can exist (other combinations are possible):

1) 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.

2) DNA-RNA. 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.

3) 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' for short.

There are two important features of hybridization:

1) 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).

2) Hybridization 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 molecules.

(Click to view Fluorescence In Situ Hybridization)

These properties 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.

These techniques 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.

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Basic Definitions

Blots are named for the target molecule.

Southern Blot
DNA cut with restriction enzymes - probed with radioactive DNA.
(Click to view an illustration of Southern Blotting)

Northern Blot
RNA - probed with radioactive DNA or RNA.

Western Blot
Protein - probed with radioactive or enzymatically-tagged antibodies.

Overview

The formation 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.

These molecules 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 molecule.

(Click to view Hybridization reactions process)

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.)

In general, the process has the following steps, detailed below:

- Gel electrophoresis
- Transfer to Solid Support
- Blocking
- Preparing the Probe
- Hybridization
- Washing
- Detection of Probe-Target Hybrids

Gel Electrophoresis

This is a technique that separates molecules on the basis of their size.

First, a 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 Electrophoresis)

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.

Note that 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 of separation)

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.

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Preparing 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.
Preparing RNA for Northern Blots: Although 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.
Preparing Proteins for Western Blots: Proteins 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 charges.

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: 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.]
Protein: 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 manner).

Sample 1 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.

Sample 2 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 sample.
(Click to view a sample result)

Different stains and staining procedures are used for different classes of macromolecules:

Staining DNA: DNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The DNA-EtBr complex fluoresces under UV light.
Staining RNA: RNA is stained with ethidium bromide (EtBr), which binds to nucleic aids. The RNA-EtBr complex fluoresces under UV light.
Staining Protein: Protein is stained with Coomassie Blue (CB). The protein-CB complex is deep blue and can be seen with visible light.

Transfer to Solid Support

After the 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.

The DNA, RNA, or protein can be transferred to nitrocellulose in one of two ways:

1) Electrophoresis, which takes advantage of the molecules' negative charge.

2) Capillary 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.

Blocking

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.

Preparing the Probe

Radioactive DNA probes for Southerns and Northerns

The objective 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 DNA fragments.

(Click to view an illustration of Microarray Technology)

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 radioactive.

4) The mixture is boiled to separate the strands and is ready for hybridization.

(Click to view an illustration of the process)

This produces a radioactive single-stranded DNA copy of both strands of the template for use as a probe.

Radioactive Antibodies for Westerns

Antibodies 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.

They are 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:

antibody-tyrosine + 125I- + H2O2 ---------> H2O + 125iodo-tyrosine-antibody

Enzyme-conjugated Antibodies for Westerns

Antibodies 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.

Hybridization

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 targets.

Washing

After hybrids 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.

Note: In 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.

Detecting 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.

Autoradiography

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.

Enzymatic Development

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 probe bound.

(Click to view the summary)

Cloning 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 for this.

Although 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.

For this 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.

1) Isolate genomic (chromosomal) DNA from human cells.

2) Create 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.

(Click to view an illustration of plasmid insertion)

3) Plate the colonies on agar plates and let them grow. These are called the master plates.

4) Press 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.

5) Break 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.

6-9) Follow 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.

10) Pick 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 gene X.

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