Southern blot
Encyclopedia
A Southern blot is a method routinely used in molecular biology
for detection of a specific DNA sequence
in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British
biologist
Edwin Southern
. Other blot
ting methods (i.e., Western blot
, Northern blot
, Eastern blot
, Southwestern blot
) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern's name. As the technique was eponym
ously named, Southern blot is capitalized as is conventional for proper noun
s. The names for other blotting methods may follow this convention, by analogy.
The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods.
Southern 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.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
for detection of a specific DNA sequence
DNA sequence
The sequence or primary structure of a nucleic acid is the composition of atoms that make up the nucleic acid and the chemical bonds that bond those atoms. Because nucleic acids, such as DNA and RNA, are unbranched polymers, this specification is equivalent to specifying the sequence of...
in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British
United Kingdom
The United Kingdom of Great Britain and Northern IrelandIn the United Kingdom and Dependencies, other languages have been officially recognised as legitimate autochthonous languages under the European Charter for Regional or Minority Languages...
biologist
Biologist
A biologist is a scientist devoted to and producing results in biology through the study of life. Typically biologists study organisms and their relationship to their environment. Biologists involved in basic research attempt to discover underlying mechanisms that govern how organisms work...
Edwin Southern
Edwin Southern
Sir Edwin Mellor Southern, FRS is an English 2005 Lasker Award-winning molecular biologist. His award was for the invention of the Southern blot, now a common laboratory procedure, when he was working at the University of Edinburgh....
. Other blot
Blot (biology)
In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier . In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane...
ting methods (i.e., Western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
, Northern blot
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
, Eastern blot
Eastern blotting
Eastern blotting is a biochemical technique used to analyze protein post translational modifications such as lipids and glycoconjugates. It is most often used to detect carbohydrate epitopes. Thus, Eastern blotting can be considered an extension of the biochemical technique of Western blotting...
, Southwestern blot
Southwestern blot
Southwestern blotting, based along the lines of Southern blotting and first described by B. Bowen and colleagues in 1980, is a lab technique which involves identifying and characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes...
) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern's name. As the technique was eponym
Eponym
An eponym is the name of a person or thing, whether real or fictitious, after which a particular place, tribe, era, discovery, or other item is named or thought to be named...
ously named, Southern blot is capitalized as is conventional for proper noun
Proper noun
A proper noun or proper name is a noun representing a unique entity , as distinguished from a common noun, which represents a class of entities —for example, city, planet, person or corporation)...
s. The names for other blotting methods may follow this convention, by analogy.
Method
- Restriction endonucleaseEndonucleaseEndonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
s are used to cut high-molecular-weight DNA strands into smaller fragments. - The DNA fragments are then electrophoresedGel electrophoresisGel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
on an agarose 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 HClHCLHCL or HCl can stand for:* Hairy cell leukemia, an uncommon and slowly progressing B cell leukemia* Hardware compatibility list...
, which depurinateDepurinationIn molecular genetics, depurination is an alteration of DNA in which the purine base is removed from the deoxyribose sugar by hydrolysis of the beta-N-glycosidic link between them. After depurination, an apurinic site is formed where the sugar phosphate backbone remains and the sugar ring has a...
s 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 hydroxide) 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, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results.
- A sheet of nitrocelluloseNitrocelluloseNitrocellulose is a highly flammable compound formed by nitrating cellulose through exposure to nitric acid or another powerful nitrating agent. When used as a propellant or low-order explosive, it is also known as guncotton...
(or, alternatively, nylonNylonNylon is a generic designation for a family of synthetic polymers known generically as polyamides, first produced on February 28, 1935, by Wallace Carothers at DuPont's research facility at the DuPont Experimental Station...
) membraneArtificial membraneAn artificial membrane, or synthetic membrane, is a synthetically created membrane which is usually intended for separation purposes in laboratory or in industry. Synthetic membranes have been successfully used for small and large-scale industrial processes since the middle of twentieth century. A...
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 suction 20X SSCSSC bufferIn biochemistry and molecular biology, the saline-sodium citrate buffer is used as a hybridization buffer, to control stringency for washing steps in protocols for Southern blotting, in situ hybridization, DNA Microarray or Northern blotting...
buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary actionCapillary actionCapillary action, or capilarity, is the ability of a liquid to flow against gravity where liquid spontanously rise in a narrow space such as between the hair of a paint-brush, in a thin tube, or in porous material such as paper or in some non-porous material such as liquified carbon fiber, or in a...
from a region of high water potentialWater potentialWater potential is the potential energy of water per unit volume relative to pure water in reference conditions. Water potential quantifies the tendency of water to move from one area to another due to osmosis, gravity, mechanical pressure, or matrix effects such as surface tension...
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 exchangeIon exchangeIon exchange is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion...
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 radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.
- The membrane is then exposed to a hybridization probeHybridization probeIn molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the 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 fluorescentFluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
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, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamideFormamideFormamide, also known as methanamide, is an amide derived from formic acid. It is a clear liquid which is miscible with water and has an ammonia-like odor. It is used primarily for manufacturing sulfa drugs and synthesizing vitamins and as a softener for paper and fiber...
, and detergents such as SDSSodium dodecyl sulfateSodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...
to reduce non-specific binding of the probe. - After hybridization, excess probe is washed from the membrane (typically using SSC bufferSSC bufferIn biochemistry and molecular biology, the saline-sodium citrate buffer is used as a hybridization buffer, to control stringency for washing steps in protocols for Southern blotting, in situ hybridization, DNA Microarray or Northern blotting...
), and the pattern of hybridization is visualized on X-rayX-rayX-radiation is a form of electromagnetic radiation. X-rays have a wavelength in the range of 0.01 to 10 nanometers, corresponding to frequencies in the range 30 petahertz to 30 exahertz and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays and longer than gamma...
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.
Result
Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe.The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods.
Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. 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.