SOUTHERN BLOT ANALYSIS

In 1975, E.M. Southern developed a method that allowed him to detect specific DNA sequences isolated from organisms of interest. This method which bears his name is called Southern blot analysis. This technique in which a labeled probe is hybridized to homologous DNA sequences is a powerful technique used in order to screen for the presence of a particular gene. First, DNA is isolated and cut with restriction endonucleases. This generates DNA fragments, which can be separated by agarose gel electrophoresis. The DNA can be transferred to a nitrocellulose membrane to immobilize it, and a specific labeled nucleic acid probe can be added. The probe hybridizes to homologous chromosomal DNA sequences based on hydrogen bonding of the nitrogenous bases. It is then possible to detect specific bands corresponding to the gene or DNA sequence of interest.

The first step is the isolation of DNA from the organism of interest. The next step is to digest the DNA using restriction endonucleases. The digested DNA can now be separated on an agarose gel. Following electrophoresis, the DNA in the agarose gel is denatured by alkali treatment to yield single-stranded DNA fragments. The gel must also be neutralized before blotting. The DNA fragments can be transferred to a nitrocellulose membrane. This transfer is by simple capillary action. The membrane is placed on the gel that sits on a sponge. Dry filter paper is placed on top of the membrane. A salt solution (sodium chloride, sodium citrate-SSC) serves as the transfer buffer and is added to the system. The buffer is attracted to the dry filter paper passing through the agarose gel and membrane in the process. The DNA in the gel is carried along with the salt solution, but becomes immobilized on the membrane. Thus, a replica of the agarose gel is created on the nitrocellulose membrane. Once the DNA is transferred, it must be fixed to the membrane in order to prevent loss of the DNA during hybridization. This is accomplished by baking the membrane at 80°C.

The next step in the process is to make and label a probe. A labeled probe will allow us to visualize where on the blot the probe has hybridized to the gene of interest (hilA). In your experiments, the probe will be made from the plasmid that contains the hilA gene. The method we will use to label our hilA gene in order to create a probe is called random primer labeling. The template (plasmid) is linearized with a restriction enzyme, denatured by boiling and then a randomly generated mixture of hexanucleotides is added along with DNA polymerase I (Klenow fragment). Denaturation will create single-stranded DNA from the double-stranded plasmid. The hexanucleotides will then pair with complementary sequences on the template (single-stranded plasmid). DNA polymerase uses the free 3'-OH ends of the primers to synthesize new regions that fill in the gaps between hexanucleotides using a supply of nucleoside triphosphates that we have added to the reaction mix. One of the nucleoside triphosphates we add is a special nucleoside triphosphate containing a digoxigenin tag. This results in the production of the probe.

The single-stranded digoxigenin labeled probe can be placed in a hybridization tube with hybridization buffer and the nitrocellulose membrane. The probe will encounter the single- stranded target DNA on the membrane by chance. If the two strands have any complementary bases, they will form base pairs thus generating a double stranded region.

The degree of homology between a probe and target DNA (gene of interest) can be estimated by performing the hybridization reactions at a particular stringency. The term stringency refers to the specificity of the hybridization reaction between target and probe. Stringency is determined by the melting temperature (Tm), the temperature at which two strands of duplex DNA are 50% dissociated. The Tm is affected by the ionic strength (amount of salt), the length of the DNA

1 strands (probe and target), the homology between probe and target, the base composition of the DNA and the concentration of probe and target relative to one another. For example, G-C base pairs containing 3 hydrogen bonds are more stable than A-T pairs containing 2 hydrogen bonds. So duplexes (double-stranded nucleic acids) that contain a high number of G-C base pairs will melt (dissociate) at higher temperatures because more energy is needed to break 3 hydrogen bonds as compared to duplex DNA that is A-T rich.

At high stringency, only completely homologous sequences will hybridize, whereas at low stringency, sequences with less than complete homology will hybridize. The hybridization reaction is performed under conditions that favor formation of hybrids, usually at temperatures well below Tm and at high salt concentrations. The post-hybridization washes determine the stringency of the experiment. Washing the blots at lower ionic strength (low salt) and at a high temperature will favor denaturation of all but the most closely homologous sequences. If hybridization is only detected at low stringency (high salt and low temperature), then the target sequence is not 100% homologous to the probe.

SOUTHERN BLOT ANALYSIS

I. Restriction Endonuclease Digestion

1. Add the proper amount of DNA (the amount depends on the particular experiment you are performing, but typically varies between 0.5-2.0 g) into a sterile microcentrifuge tube (the volume can be between 0.5 - 18 l).

2. Add sterile dH2O so that the total volume is 18 l.

3. Add 2 l of 10X restriction endonuclease buffer, mix well and centrifuge for 5 seconds.

4. Heat the samples at 65°C for 5 minutes.

5. Keep the tubes on ice.

6. Add 0.5 l of the proper restriction endonuclease, mix well and centrifuge for 5 seconds.

7. Incubate the tubes at 37°C for 1 hour.

8. Store all tubes at –20°C until needed.

II. Agarose Gel Electrophoresis

1. Make a 1.0 % agarose gel in 1X TAE buffer.

50X Tris-acetate buffer (for 500 mL)

a. 2.0M Tris – 121g b. 28.6 mL of glacial acetic acid c. 50 mL of 0.5M EDTA (pH 8.0) - 9.3g EDTA/50ml d. Adjust pH to 8.0

2 e. autoclave

2. After mixing the agarose and buffer, place the flask onto a ring stand and gently swirl the flask while heating the agarose solution over a Bunsen burner. Bring the solution to a boil and remove the flask from the flame. BE CAREFUL NOT TO LET THE AGAROSE SOLUTION BOIL OUT OF THE FLASK.

3. Allow the agarose to cool to 65°C and add 0.5 g/mL of ethidium bromide (our stock is 10 mg/mL ethidium bromide solution) and gently mix. WEAR GLOVES FROM THIS POINT ON BECAUSE ETHIDIUM BROMIDE IS A POWERFUL MUTAGEN! Dispose of the pipette tip only in waste containers specifically marked for ethidium bromide.

4. Pour the agarose into the gel tray (make sure the side supports are in place and the gel comb has been inserted into the tray). Pour the agarose solution into the gel tray. Allow the gel to harden for about 20-30 minutes.

5. Retrieve the restriction digests you plan to run. Heat your samples at 65°C for 5 minutes. Pipette 3 l of sterile tracking dye into each sample. Mix each sample well and centrifuge the samples in a microcentrifuge for 5 seconds. Place the tubes back on ice.

6. Make up enough 1X Tris acetate buffer in order to fill the electrophoretic chamber. After your gel has hardened, gently remove the comb and the side supports from the gel. Place the gel into the chamber and add enough 1X Tris acetate buffer to cover the gel.

7. Carefully load your samples into the gel using a micropipette. Be sure to record what wells contain your samples.

8. Place the cover onto the electrophoretic chamber and connect the electrodes to the power supply. Run the gel at about 60-80 volts until the tracking dye has migrated more than half way through the agarose gel.

9. View the gel under an ultraviolet light and take a photograph. Be sure to record the position of all DNA bands that appear in the gel.

10. Following electrophoresis, denature the gel by incubating in 0.5 M NaOH, 1 M NaCl two times for 30 minutes each.

11. Neutralize the gel with 0.5 M Tris-HCl pH 7.5, 3 M NaCl two times for 30 minutes each.

III. Southern Blot

1. The DNA can be transferred from the agarose gel to the nitrocellulose membrane following neutralization.

2. The blotting apparatus can be assembled during the neutralization process. The blotting buffer is sterile 10X SSC (salt, sodium citrate).

3 20X SSC buffer (salt, sodium citrate) 3.0 M NaCl (M.W.=58.44) 0.3 M sodium citrate (M.W.=294.1) Add the above chemicals to 800 mL of dH2O. Adjust the pH to 7.0 and bring the volume to 1 liter. Autoclave

3. The nitrocellulose membrane should never be touched with your hands. Be sure to wear gloves. After cutting the membrane to the size of the gel, soak the membrane in sterile dH2O for 30 seconds followed by 15 minutes in 10X SSC. When placing the nitrocellulose membrane on the gel care should be taken to remove all air bubbles.

4. Transfer of DNA is completed in 15-20 hours. Following blotting, the lanes of the gel are marked on the nitrocellulose and the membrane is soaked in 2X SSC for about 10 minutes. The nitrocellulose membrane is then place between a few sheets of filter paper, wrapped in foil and baked in an oven at 80°C for 2 hours. This will cross-link the DNA to the nitrocellulose membrane. The membrane can then be stored at room temperature.

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