We separate the DNA to be tested from the rest of the cellular material in the nucleus.The probes are labeled with a marker and complementary to the target DNA as a result we can detect one molecule of target in a mixture of millions after hybridization as the reactions are specific. Hybridization is a technique in which a double stranded DNA molecule is formed in between a single stranded DNA probe and a target single stranded DNA. It is based on the principle of transfer of separated DNA fragments to a carrier membrane (usually nitrocellulose) using gel electrophoresis and subsequent identification of specific DNA fragmentsby labelled probe hybridization. The most common and popular membranes are made of nitrocellulose, uncharged nylon positively charged nylon but they are interchangeable depending on the applications. There are different types of membrane, transfer buffer and transfer methods to set up a southern blot. The DNA are then exposed to hybridization analysis allowing bands with sequence resemblance to a labeled probe to be identified. During southern blotting, the DNA fragments are immobilized as a result, the membrane carries a semi-permanent reproduction of the banding pattern of the gel. Southern blotting is a molecular biology technique used for DNA detection, characterization, and quantification.Īn example of RFLP(restriction fragment length polymorphism), southern blotting can be defined as an analytical technique for identifying specific sequences of DNA by separating fragments on a gel and transferring them to a second medium (carrier membrane) on which hybridization testing may be carried out. Southern integrated three innovations to create the Southern blot – restriction endonucleases, gel electrophoresis and blotting through methods.DNA fragments were differentiated using electrophoresis based on size, then transferred to a membrane and hybridized with a radio labeled DNA probe. It was introduced as a technique to detect particular sequence of DNA in DNA samples. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.Edwin Southern, the inventor of Southern blotting started a trend to his invention after him. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. A second alternate protocol describes a transfer method based on a different transfer-stack setup. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. 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.
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