A test commonly used in molecular biology and genetics, the purpose of the test being to check for a match between dna molecules. Southern blotting of genomic dna howard judelson 601 1. Southern who developed this procedure at edinburgh university in the 1970s. Dna is applied to an agarose gel, and electrophoresis separates the fragments of dna according to size. Each sample of dna has been digested with the same restriction enzyme ecori. Interpretation of the southern blot microsatellite. Neal burnette and is a play on the name southern blot, a technique for dna detection developed earlier by edwin southern. Southern blotting is a technique of biotechnology which is used to determine and isolate the particular gene or dna sample present in the genome. The very long pieces of chromosomal dna are cleaved into much smaller units that range from a few hundred to tens of.
The dna appears smeared, with no discrete bands, as no one dna sequence is present in more than one copy. Try pulling your boss up on that one next time he is in midflow talking about a northern blot in a departmental presentation. Thus, eastern blot can be considered an extension of the biochemical technique of western blot. The northern blot technique was developed in 1977 by james alwine, david kemp, and george stark at stanford university, with contributions from gerhard heinrich.
Southern blot analysis reveals information about dna identity, size, and abundance. Southern blotting and secondary article related dna. Determines the copy number of a gene within the genome of an organism. Southern blotting is the transfer of dna fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the dna fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. Southern blotting this part of the protocol has to be carried out at a postpcr lab note.
Southern blotting combines transfer of electrophoresisseparated dna fragments to a filter membrane and subsequent fragment detection by probe hybridization the method is named after the british biologist edwin southern, who first published it in 1975. Southern blot biomolecules mcat khan academy youtube. A southern blot is a method used in molecular biology for detection of a specific dna sequence in dna samples. Biomedical and biological sciences 20,932 views 17. Southern blot definition of southern blot by merriamwebster. The purpose of each technique may be different, but all share the same principle and methodology, with a few minor deviations and modifications. Dna fragments are separated by electrophoresis on an agarose gel, transferred blotted onto a nitrocellulose or nylon membrane, and hybridized with complementary labeled nucleic acid probes. Southern blotting is used in case of dna, northern blotting in case of rna, western blotting in case of proteins, and eastern blotting in case of posttranslational modifications of proteins. Different blotting techniques are used to identify unique proteins and nucleic acid sequences. Current protocols in protein science, 01 may 2001, appendix 4. Southern blotting definition of southern blotting by. Mutation detection by southern blotting article in methods in molecular biology clifton, n. Summary agarose gel 12 3 dna markers restricted dna buffer wick support gel paper towels nylon membrane nylon membrane figure 1.
Southern blot analysis definition of southern blot. Northern blotting takes its name from its similarity to the first blotting technique, the southern blot, named for biologist edwin southern. The dna detected can be a single gene, or it can be part of a larger piece of dna such as a viral genome. Terms in this set 11 southern blottingused to detect a particular gene sequence within a mixture of many chromosomal dna fragments. As a result subsequent blotting techniques have used. The digested dna was subjected to southern blot analysis as described in table 1. In my southern blot, i found only smears, not the desired. Southern blotting brown 1993 current protocols in molecular. After the electrophoresis the separated dna fragments are denaturated and transferred to a nitrocellulose or.
Place the dna southern blot into a small hybridization tube by carefully rolling it with the dna side toward the inside of the. This protocol describes a basic method to perform the southern blot. Extract and purify the dnarna from either cells or tissue. Recognition will depend on the probe and on the hybridization conditions, such as ionic. Southern blotting is a hybridization technique for identification of particular size of dna from the mixture of other similar molecules. Dna was isolated from cultures of rat liver epithelial cells. Southern blotting is designed to locate a particular sequence of dna within a complex mixture. It allows investigators to determine the molecular weight of a restriction fragment, to measure relative amounts in different samples and to locate a particular sequence of dna within a complex mixture. The southern blot is then set up,as illustrated in figure 1,with a highsalt transfer bu. What is the major difference between northern and southern blotting. Southern vs northern vs western blotting techniques lab manager. Hybridization, southern blot, northern blot, and microarrays.
The southern blot is used to detect the presence of a particular dna fragment in a sample. Southern was the scientist who developed the technique of southern blotting in 1970. Can anyone explain major difference between northern and southern blotting. Southern blotting principle, procedure and application. Southern, who developed the technique in edinburgh, scotland in the 1970s. Southern blotting is the transfer of dna fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the dna fragments, so the. Southern blot hybridization refers to the detection of specific dna fragments that have been separated by gel electrophoresis figure 1. Department of agronomy southern blot 4 updated 031405 9. Mutation detection by southern blotting request pdf. Southern who developed this procedure at edinburgh university in the 1975. Set the uv transilluminator to auto and press start. The name western blot was given to the technique by w.
In the southern blot technique, dna fragments are separated by a method called agarose gel electrophoresis, transferred blotted onto membrane filters, and hybridized with complementary radiolabeled probes. The eastern blot, or eastern blotting, is a biochemical technique used to analyze protein posttranslational modifications ptm including the addition of lipids, phosphates, and glycoconjugates. A process used for dna analysis developed by edwin southern in 1975 is also referred to as dna blotting edwin southern. The gel is then placed atop a thin sponge wick resting in a dish of salt solution, and a special filter typically nitrocellulose is placed on top of the gel.
In a southern blot, genomic dna is digested with a restriction enzyme that cuts at a short specific 48 bp segment of dna fig. To oversimplify, dna molecules are transferred from an agarose gel onto a membrane. Southern blotting is an example of rflp restriction fragment length polymorphism. This article also offers a southern blot protocol and a northern blot protocol. It has great value when it is necessary to discriminate between antibodies produced in response to vaccination and those produced as a result of infection. The southern blot is used to detect the amount of dna in a mixture. Southern blot, named for the man who first developed it, edward southern. The southern blot is use to verify the presence or absence of a specific nucleotide sequence in dna from different sources. Blot membrane in filter paper and crosslink still inside the filter paper. The left panel is an electrophoretic gel stained with ethidium bromide. Once the agarose gel has run long enough for the desired fragment separation to be obtained, photograph the gel using a ruler to. Analysis of human mitochondrial dna content by southern blotting. Southern blot definition is a blot consisting of a nitrocellulose or nylon sheet containing spots of dna for identification by a suitable molecular probe. Analysis of human mitochondrial dna content by southern blotting and nonradioactive probe hybridization.
With northern blotting, rna molecules are transferred and with western blotting, protein molecules are transferred. In my southern blot, i found only smears, not the desired band picture attached. Just as with protein and rna, the dna of a cell can be released when that cell is broken open. Get a printable copy pdf file of the complete article 882k, or click on a page image below to browse page by page. It is most often used to detect carbohydrate epitopes. Comparison of southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus dna. Southern blotting 2 capillary blottingfragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action. Abstract southern blotting is the transfer of dna fragments from an electrophoresis gel to a membrane support the properties and advantages of the different. Southern blotting and related dna detection techniques.
Soak the remaining gel in 1x tae with ethidium bromide for 10 min and take a picture. Southern blotting was introduced by edwin southern in 1975 as a method to detect specific. Blotting allows the detection of specific molecules among a mixture separated by gel. Southern and northern blotting protocols involve the following major steps. However, because of its unpopular nature finding someone that can teach you about how to do a good northern blot can be hard. Perhaps only one fragment will be recognized although the same two fragments are still being formed. A method developed by em southern for detecting and manipulating specific dna sequences previously separated by gel electrophoresis using a radioactively or chemicallylabeled dna probe with sequence similarity. The southern blot is used to detect a specific dna sequence in dna samples.
What is the major difference between northern and southern. Southern blotting is a method used to tran sfer dna from an agarose gel to a membrane, where the dna can be subsequently probed fo r a specific sequence. Note that among these techniques, only the southern blot should be capitalised since it refers to southerns name, the others nothern, western etc are not proper nouns, so should not be capitalised. Comparison of southern blot hybridization and polymerase chain. Southern blotting is the transfer of dna fragments from an electrophoresis gel to. It is a classic technique that involves separating dna fragments based on size via electrophoresis, transferring them to a membrane, hybridization with a labeled sequencespecific probe, washing, and finally detection of labeled dna bands. Southern blot analysis was one of the first techniques to find wide clinical application. Southern blotting is the original blotting technique, which started the naming system. A southern blot assay for detection of hepatitis b virus covalently. Southern blots are a method of detecting dna and named after the surname of their inventor ed southern.
774 1519 536 917 1253 1061 1412 1013 602 831 184 1093 210 729 701 565 944 746 1519 1312 1143 8 1444 1619 556 235 778 1025 1242 1354 914 959 1075 1308 1147 1323 1258 167 923 1160 1282 1055 613 479 756