Understanding the Southern Blot Technique

Southern blot is a classic technique in molecular biology that reveals information about DNA identity, size, and abundance. And it can be used for detection of a specific DNA sequence in DNA samples. The first step in southern blotting is the digestion of the DNA samples with an appropriate restriction enzyme. A restriction enzyme also called restriction endonuclease, is an enzyme that cleaves DNA into fragments, at or near, specific recognition sites, within molecules known as restriction sites. Once the restriction enzyme is added, the samples are incubated at 37°C overnight. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. Therefore, Various DNA fragments of different sizes are obtained. Then, the DNA fragments produced by restriction endonuclease digestion are separated by gel electrophoresis. For gel electrophoresis separation, a loading buffer is added to the DNA samples, and it is used as a tracking dye, which migrates in the same direction as DNA, allowing the user to monitor the progress of the separation. Agarose gel electrophoresis is most commonly used to separate mixtures of DNA fragments of varying sizes. During electrophoresis, a molecular weight size marker known as a DNA ladder is commonly used to determine the size of DNA fragments in the samples. The DNA ladder is added into well at one end of the gel. Once the molecular weight size marker is added, the DNA samples are loaded into wells. Then an electric current is applied to pull the samples through the gel. Based on their charge and size, the molecules will travel through the gel at different speeds, allowing them to be separated from one another. The phosphate backbone of the DNA molecule is negatively charged. Therefore, when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. When the gel is stained with an intercalating dye such as ethidium bromide, the DNA fragments can be seen under UV light as bands. each representing a group of same-size DNA fragments. After the separation of the DNA molecules, the double-stranded DNA fragments are denatured with an alkaline solution consisting of sodium hydroxide. The gel is soaked in the alkaline solution with gentle agitation. At an alkaline pH, hydroxide ions are predominant, consequently, guanine and thymine will be deprotonated. and exist as negatively charged conjugate bases. This process breaks the hydrogen bonds between the oligonucleotides, causing the separation of the two DNA strands. After the denaturation treatment, the alkaline solution is removed. Then, a neutralizing solution is used to neutralize the pH of the gel. This allows for a more efficient transfer of DNA in southern transfers. While the gel is neutralizing, filter paper and membrane are prepared for southern blotting. To transfer the DNA fragments to the membrane, a transfer buffer, a solid support, and a sheet of blotting paper acts as a wick for the transfer solution are used. The wick is placed over the solid support in the transfer reservoir, so the ends will be in the transfer buffer. Then, it is wetted with the transfer solution. Next. pieces of extra thick blotting paper are placed on top of the wick. Then, they are wetted with the transfer solution. Once the gel is neutralized, it is placed on the thoroughly wetted wicking paper. Then, a sheet of nylon or nitrocellulose membrane with the same size as the gel is pre-wetted with the transfer solution and placed on top of the gel. Next, pre-wetted pieces of extra-thick blotting paper are placed on top of the membrane. The exposed areas of the wick are covered with strips of plastic wrap to prevent transfer buffer from bypassing the gel during the transfer process. Finally, a dry stack of paper towels is placed on top of the membrane and gel, then a glass plate is placed on top of these sheets paper with a weight to maintain tight contact between the gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential is then used to move the DNA from the gel onto the membrane. Consequently, ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. The transfer is allowed to proceed overnight, then once it is completed, the blotting material and membrane are carefully removed from the gel. Next. the membrane is briefly rinsed to remove any agarose that may be stuck during the transfer. Then, it is exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane. After attachment of the DNA fragments to the membrane, hybridization with radiolabel DNA probes is performed. The membrane is placed in a bottle containing a pre-hybridization solution. which is used to reduce nonspecific hybridization with the probe. Next, the bottle is incubated in hybridization oven at 42°C for 2 hours. Once the incubation is complete, the pre-hybridization solution is removed. Then, a hybridization buffer is added into the bottle. Next, labeled DNA probes are added to the hybridization solution. The probes are fragments of DNA of variable length, which can be radioactively or fluorescently labeled. Once the hybridization probes are added, the bottle is incubated overnight in the hybridization oven at 42°C. DNA contains a large quantity of phosphorus in the phosphodiester linkages between bases in the oligonucleotide chain. DNA can therefore be tracked by replacing its non-radioactive phosphorus with radioactive phosphorus-32. The radioactively labeled DNA probes hybridize to their complementary sequences in the DNA fragments. After the hybridization of each probe to its target sequence, the hybridization solution is removed. Next, a wash buffer is added into the bottle, then the membrane is incubated at 52°C for 30 minutes. The washing process is repeated three times to remove unbound and weakly binding probe. After hybridization, an autoradiography method is carried out to identify the location of radioactively labeled DNA in the membrane. The southern blot filter is placed inside a light-proof cassette box. Then, an X-ray film is laid over the top. The cassette is closed and left for several hours to several days. The radioisotope-labeled DNA exposes the film, which when developed shows a pattern of black bands that indicate the positions of labeled DNA in the blot membrane. Subsequently, these informations can be used to determine which gene is present in each sample. Also, the position of each gene can be identified in the gel. Consequently, after electrophoresis, bands of interest can be cut out from the gel so that each gene can be isolated for further analysis.

A restriction enzyme also called restriction endonuclease, is an enzyme that cleaves DNA into fragments, at or near, specific recognition sites, within molecules known as restriction sites. Once the restriction enzyme is added, the samples are incubated at 37°C overnight. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA.

Therefore, Various DNA fragments of different sizes are obtained. Then, the DNA fragments produced by restriction endonuclease digestion are separated by gel electrophoresis. For gel electrophoresis separation, a loading buffer is added to the DNA samples, and it is used as a tracking dye, which migrates in the same direction as DNA, allowing the user to monitor the progress of the separation.

Agarose gel electrophoresis is most commonly used to separate mixtures of DNA fragments of varying sizes. During electrophoresis, a molecular weight size marker known as a DNA ladder is commonly used to determine the size of DNA fragments in the samples. The DNA ladder is added into well at one end of the gel. Once the molecular weight size marker is added, the DNA samples are loaded into wells. Then an electric current is applied to pull the samples through the gel.

Based on their charge and size, the molecules will travel through the gel at different speeds, allowing them to be separated from one another. The phosphate backbone of the DNA molecule is negatively charged. Therefore, when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.

When the gel is stained with an intercalating dye such as ethidium bromide, the DNA fragments can be seen under UV light as bands. each representing a group of same-size DNA fragments. After the separation of the DNA molecules, the double-stranded DNA fragments are denatured with an alkaline solution consisting of sodium hydroxide.

The gel is soaked in the alkaline solution with gentle agitation. At an alkaline pH, hydroxide ions are predominant, consequently, guanine and thymine will be deprotonated. and exist as negatively charged conjugate bases. This process breaks the hydrogen bonds between the oligonucleotides, causing the separation of the two DNA strands.

After the denaturation treatment, the alkaline solution is removed. Then, a neutralizing solution is used to neutralize the pH of the gel. This allows for a more efficient transfer of DNA in southern transfers.

While the gel is neutralizing, filter paper and membrane are prepared for southern blotting. To transfer the DNA fragments to the membrane, a transfer buffer, a solid support, and a sheet of blotting paper acts as a wick for the transfer solution are used. The wick is placed over the solid support in the transfer reservoir, so the ends will be in the transfer buffer. Then, it is wetted with the transfer solution.

Next. pieces of extra thick blotting paper are placed on top of the wick. Then, they are wetted with the transfer solution.

Once the gel is neutralized, it is placed on the thoroughly wetted wicking paper. Then, a sheet of nylon or nitrocellulose membrane with the same size as the gel is pre-wetted with the transfer solution and placed on top of the gel. Next, pre-wetted pieces of extra-thick blotting paper are placed on top of the membrane. The exposed areas of the wick are covered with strips of plastic wrap to prevent transfer buffer from bypassing the gel during the transfer process.

Finally, a dry stack of paper towels is placed on top of the membrane and gel, then a glass plate is placed on top of these sheets paper with a weight to maintain tight contact between the gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential is then used to move the DNA from the gel onto the membrane. Consequently, ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. The transfer is allowed to proceed overnight, then once it is completed, the blotting material and membrane are carefully removed from the gel. Next.

the membrane is briefly rinsed to remove any agarose that may be stuck during the transfer. Then, it is exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane. After attachment of the DNA fragments to the membrane, hybridization with radiolabel DNA probes is performed.

The membrane is placed in a bottle containing a pre-hybridization solution. which is used to reduce nonspecific hybridization with the probe. Next, the bottle is incubated in hybridization oven at 42°C for 2 hours. Once the incubation is complete, the pre-hybridization solution is removed. Then, a hybridization buffer is added into the bottle.

Next, labeled DNA probes are added to the hybridization solution. The probes are fragments of DNA of variable length, which can be radioactively or fluorescently labeled. Once the hybridization probes are added, the bottle is incubated overnight in the hybridization oven at 42°C.

DNA contains a large quantity of phosphorus in the phosphodiester linkages between bases in the oligonucleotide chain. DNA can therefore be tracked by replacing its non-radioactive phosphorus with radioactive phosphorus-32. The radioactively labeled DNA probes hybridize to their complementary sequences in the DNA fragments.

After the hybridization of each probe to its target sequence, the hybridization solution is removed. Next, a wash buffer is added into the bottle, then the membrane is incubated at 52°C for 30 minutes. The washing process is repeated three times to remove unbound and weakly binding probe. After hybridization, an autoradiography method is carried out to identify the location of radioactively labeled DNA in the membrane. The southern blot filter is placed inside a light-proof cassette box.

Then, an X-ray film is laid over the top. The cassette is closed and left for several hours to several days. The radioisotope-labeled DNA exposes the film, which when developed shows a pattern of black bands that indicate the positions of labeled DNA in the blot membrane. Subsequently, these informations can be used to determine which gene is present in each sample.

Also, the position of each gene can be identified in the gel. Consequently, after electrophoresis, bands of interest can be cut out from the gel so that each gene can be isolated for further analysis.

Transcript for:Understanding the Southern Blot Technique

Transcript for:
Understanding the Southern Blot Technique