Nucleic Acid Hybridizations
The hybridization of a radioactive probe to filter bound DNA or RNA is one of the most informative experiments that is performed in molecular genetics. Two basic types of hybridizations are possible.
Southern hybridization - hybridization of a probe to filter bound DNA; the DNA is typically transferred to the filter from a gel
Northern hybridization - hybridization of a probe to filter bound RNA; the RNA is typically transferred to the filter from a gel
Probes are the primary tool used to identify complementary sequences of interest. Generally, the probe is a clone developed by inserting DNA into a vector. Most often these are plasmid clones.
Probe - a single-stranded nucleic
acid that has been radiolabelled and is used to identify a complimentary
The hyrbridization process involves two different steps. First the nucleic acid must be immobilized on a filter. This is generally called a "Southern Transfer" procedure. The second step is the actual hybridization of the probe to the filter bound nucleic acid.
The following steps describe the Southern transfer procedure.
Southern hybridizations with plant DNA is not a trivial matter. The primary requirement for a successful experiment is that the DNA to be probed is digested to completion. We have already discussed the choice of enzymes in this regard. Even when using compatible enzymes (not GC or GXC sensitive) monitoring the completeness of the reaction is essential for consistent results.
Once you are satisfied that you completely digested the DNA and are confident that it was successfully transferred to the filter membrane, the next step is perform the actual hybridization. The following steps describe the procedure.
Steps in Southern Hybridization Procedure
The temperature and salt concentrations at which we perform a hybridization has a direct effect upon the results that are obtained. Specifically, you can set the conditions up so that your hybridizations only occur between the probe and a filter bound nucleic acid that is highly homologous to that probe. You can also adjust the conditions the hybridzation is to a nucleic acid that has a lower degree of homology to the probe.
Your hybridization results are directly
related to the number of degrees below the Tm of DNA at which the
experiment is performed. For a aqueous solution
of DNA (no salt) the formula for Tm is:
Tm = 69.3oC + 0.41(% G + C)oC
Tm = 69.3oC + 0.41(% G + C)oC
From this formula you can see that the
GC content has a direct effect on Tm. The following examples,
demonstrate the point.
Tm = 69.3oC + 0.41(45)oC = 87.5oC (for
wheat germ) Tm = 69.3oC + 0.41(40)oC = 85.7oC Tm = 69.3oC + 0.41(60)oC = 93.9oC
Tm = 69.3oC + 0.41(45)oC = 87.5oC (for wheat germ)
Tm = 69.3oC + 0.41(40)oC = 85.7oC
Tm = 69.3oC + 0.41(60)oC = 93.9oC
Hybridizations though are always performed
with salt. This requires another formula which that takes the salt
Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) - 0.72(% formamide)
The salt solution that is most often used in hybridization experiments is SSC (standard sodium citrate). Different concentrations of this solution are used at different steps in the hybridization procedure. The following table gives the Na+ concentration for different strengths of SSC. Remember that this value is essential to derive the Eff Tm.
Na+ ion concentration of different strengthsof SSC
Another relevant relationship is a that 1% mismatch of two DNAs lowers the Tm 1.4oC. So in a hybridization with wheat germ that is performed at Tm - 20oC (=67.5oC), the two DNAs must be 85.7% homologous for the hybridization to occur. 100% - (20oC/1.4oC) = 85.7% homology
Let's now look at an actual experiment, the hybridization of a probe with filter bound wheat DNA in 5X SSC at 65oC. The first step is to derive the Eff Tm.
Eff Tm = 81.5 + 16.6(log 0.825) + 18.5 = 98.6oC
These types of hybridization experiments
are typically performed at Tm - 20oC. A typical temperature of
100 - [(98.6-65.0)/1.4] = 100 - (23.6/1.4) = 83.1%.
The next step in a hybridization experiment is to wash the filter. This is normally done in two steps. First a non-stringent wash is performed to remove the non-specifically bound DNA and the second wash is performed at a higher stringency that only permits highly homologous sequences to remain bound to the filter. Controlling the stringency is an important step in these experiments.
Stringency - a term used in hybridization experiments to denote the degree of homology between the probe and the filter bound nucleic acid; the higher the stringency, the higher percent homology between the probe and filter bound nucleic acid
Non-stringent wash: normally 2X SSC, 65oC
Eff Tm = 81.5 + 16.6[log(0.33)] + 0.41(45%)= 92.0oC
%Homology = 100 - [(92-65)/1.4] = 80.7%
Stringent wash: normally 0.1X SSC, 65oC
Eff Tm = 81.5 + 16.6[log(0.0165)] + 0.41(45%) = 70.4oC
%Homology = 100 -[(70.4-65)/1.4] = 96.1%
This example shows that the final wash is the one of concern when determining the relatedness of the probe and the filter bound nucleic acid.
An example: Bowman-Kirk Protease Inhibitor Final wash is performed at 0.2X SSC, 55oC; assume 45% GCcontent
Eff Tm = 81.5 + 16.6[log(0.033)] + 18.5 = 75.4oC
%Homology = 100 - [(75.4-55.0)/1.4] = 85.4%
The point to this last example to emphasize that your percent homology is directly related to your most stringent condition in your hybridization experiment. This invariably is the final wash. Thus, you only need to make this calculation to determine the stringency of your experiment.
What You Can Learn from Southern Hybridizations
Southern hybridizations have many applications. The first application after cloning a gene is often to determine how many copies of the gene are in the species from which the gene was cloned. This experiment is performed by hybridizing a clone of the gene to total DNA that has been digested with several enzymes. The procedure is termed a genomic southern.
One gene that has drawn intense interest because of its potential applied usage in plant biotechnology is chitinase. We have already discussed the isolation of a clone for this gene from bean. As you can imagine, the gene has also been cloned from other species. The first page of hybridization handout shows the southern hybridization pattern obtained from cucumber, rice and bean. These hybridizations show that these species contain different copy numbers for the gene.
A second application for southern hybridizations
is the estimation of copy number of a specific gene. This experiment is
performed by running several lanes with different copy numbers of the gene
to which you are
Southern hybridization analysis can also
be performed to determine if a phenotypic mutation is due to a structural
change in the gene controlling the trait of interest. If a gene undergoes
an insertion or deletion the resulting
hybridization pattern would be changed. Insertional mutagenesis would generate
fragments of an increased size whereas
deletions would reduce the size of the hybridizing band. Two tomato mutants,
Neverripe (nr) and ripening inhibitor (ri)
express polygalacturonase, an enzyme involved in fruit ripening, at
Copyright © 1998. Phillip McClean