SOUTHERN BLOTTING : E.M SOUTHERN develop southern blotting in 1975. It is used to analyse the related genes in DNA restriction fragements. It also provides the physical map of restriction sites in a particular gene which is located on chromosomes, southern blotting can also gives us information about the number of copies gene in a particular genome,most important it gives us information about degree of similarity of the genes when compared with the other complementry genes.
PROCEDURE OF SOUTHERN BLOTTING :Restriction endonuclease enzyme are used to cut high-molecular-weight DNA strands into smaller fragments.
The DNA fragments of unequal length are then electrophorsed on an agrose gel to separate them by size.
If some of the DNA fragments are larger than 15 kb then prior to blotting, the gel may be treated with an acid, such as dilute HCL, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.
If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydrooxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, single DNA strands are also better for for later hybridization to the probe, and destroys any residual RNA that may still be present in the DNA.
A sheet of nitrocellulose membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.
The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet ray (in case of nylon membrane) to permanently attach the transferred DNA to the membrane.
The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluroscent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA.
After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.
APPLICATION OF SOUTHERN BLOTSouthern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.
PROCEDURE OF SOUTHERN BLOTTING :Restriction endonuclease enzyme are used to cut high-molecular-weight DNA strands into smaller fragments.
The DNA fragments of unequal length are then electrophorsed on an agrose gel to separate them by size.
If some of the DNA fragments are larger than 15 kb then prior to blotting, the gel may be treated with an acid, such as dilute HCL, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.
If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydrooxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, single DNA strands are also better for for later hybridization to the probe, and destroys any residual RNA that may still be present in the DNA.
A sheet of nitrocellulose membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.
The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet ray (in case of nylon membrane) to permanently attach the transferred DNA to the membrane.
The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluroscent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA.
After hybridization, excess probe is washed from the membrane, and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.
APPLICATION OF SOUTHERN BLOTSouthern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.
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