Southern blot procedure

A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure. 

RFLP analysis by the Southern blot procedure and detection with probe specific for each VNTR are the basis of DNA fingerprinting. Using just four DNA probes, paternity can be excluded with a cumulative probability of 99.9% (J2). 

FIGURE 1 The Southern blot procedure, as applied to DNA fingerprinting. This procedure was named after Jeremy Southern, who developed the technique. 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

The electrotransfer of proteins onto (non-specific) binding membranous sheets is named Western hlot in contrast to the transfer of DNA (Southern hlot) and of RNA (Northern hlot). The main advantage of blotting procedures lies in the immobilization and presentation of macromolecules on the surface of a solid planar material. This presentation leads to an easy access of reactants in the opposite to the diffusion-controlled motion of reaction partners within gels or macroporous spheres. 

Two types of screening procedures are used to identify ES cell clones carrying a targeted integration of the construct DNA polymerase chain reaction (PCR) and Southern blot analysis (Southern, 1975). Both methods rely upon the specific juxtaposition of vector components and target locus sequences after homologous recombination. 

El 1. The name Western blot is derived from other blotting procedures called the Southern blot (developed by Earl Southern) and the Northern blot. What are the differences among these three types of blotting techniques and what is the purpose of each ... 

Fig. 1. Southern blotting. The procedure shown is the original method of Southern using capillary action to blot the DNA bands from the gel to the nitrocellulose membrane. Electrolytic transfer is now often used instead.

Northern blotting follows much the same procedure as Southern blotting except that the sample analyzed by gel electrophoresis and then bound to the filter is RNA not DNA. Therefore the technique detects RNA molecules that are complementary to the DNA probe. If cellular RNA is electrophoresed, for example, a DNA probe for a specific mRNA could be used to detect whether that mRNA was present in the sample. The migration distance of the RNA in the gel would also allow estimation of its size. Note that Southern blotting (for DNA) obtained its name after its inventor (E. Southern) the name Northern blotting (for RNA) was devised later and is a geographical pun ... 

Much of the methodology for DNA preparation, and Southern blot analysis used for DNA fingerprinting, is pubhshed elsewhere (Honma et al., 1993) and the reader should refer to this for further details (particularly for steps 1-3 above) and to the procedures supplied with the Jeffreys multilocus probes). 

In this chapter, procedures are described for the enrichment and isolation of CCC DNA and total DNA from both cultured PDH and fresh and frozen liver (or other organ) specimens. The technique for the former is essentially a modified Hirt (6) high-salt extraction (5), Detection of the replicative intermediates is by Southern blot hybridization analysis, which can be performed using... 

For qualitative detection, 20 pL of the PCR reaction product is electrophore-sed in 1.8% agarose gels, and Southern blot hybridized to a 32P-end-labeled internal probe (EmcPl) following standard procedures. Blots can be exposed for 3-24 h. Alternatively, a nonradioactive ELISA-based detection system may be used as described in Subheading 3.4. 

Northern blot. A gel-based laboratory procedure that locates on a gel mRNA sequences that are complementary to a piece of DNA used as a probe. Named after Dr. E. M. Southern, who invented the Southern blot, it was adapted to RNA and named the Northern blot. 

The Southern blot (named for its inventor, E. M. Southern) is a method for hybridizing one or more labeled DNA probes to a large number of DNA fragments and discriminating among them. The procedure depends on the ability of denatured DNA single strands to bind tightly to nitrocellulose under certain conditions. 

Procedure for a Southern blot. This analysis allows detection of a specific fragment of DNA by hybridization of a radioactive probe with a complementary sequence. 

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