This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Investigators routinely employ a manual approach to probe design. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.Īutomated design of genomic Southern blot probesīackground Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. 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. The downward capillary method described in The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols 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. The method can also be used with neutral nylon membranes but less DNA will be retained. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), 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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