Southern blot protocol

Blot the proteins onto the affinity membrane either by dot blot (Protocol 4.8), by electrotransfer (Protocol 2.5.3), or by capillary blotting (handling analogously to Southern blotting Protocol 2.5.9). 

Since the late 1970s, Southern blot analysis has had greater applicability in linkage analysis and detection of acquired chromosomal rearrangements. Currently, this technique forms the basis of a majority of protocols used in the biopsy... 

Gl. Gold, B., Radu, D., et al., Diagnosis of fragile X syndrome by Southern blot hybridization using a chemiluminescent probe A laboratory protocol. Mol. Diagn. 5(3), 169-178 (2000). 

Some of the above-mentioned methods have limitations. Southern blot analysis requires a large amount of high-quality DNA, and the results are unreliable if the sample has a low percentage of tumor cells (Ross and Fletcher, 1998). For these and other drawbacks, this method is precluded from becoming a routine clinical protocol to determine HER-2 status. 

When in vivo experiments in infected adult ducks are performed, the methods described for the isolation of DHBV DNAs from cell culture material are modified slightly to accommodate a larger volume of cellular material. Although the protocol here describes the extraction of liver tissue, most tissues could be successfully extracted using this method. The DNA isolated using these methods may then be analyzed by Southern blot hybridization. 

The RAPD protocol is relatively quick and easy, fluorescence is used in lieu of radioactivity, and the assay saves time, effort, and the myriad risks and precautions associated with Southern blot technology. 

Although Southern blots are usually prepared using capillary or vacuum techniques, we have found that the following protocol gives comparable results and is much more convenient to set up. 

To generate a PCR-STR probe, e qitimized reaction and cycling conditions are used to evenly produce strands over a broad size range, and strands of 300-700 bp are then excised and recovered fi om an agarose gel. A fiaction of the recovered DNA is then used in a one-sided PCR reaction to produce -labeled probes. These labeled PCR-STR probes are used iudividually in Southern blot hybridization protocols. Using longer repetitive DNA probes may yield more stable, clearer results than obtained under similar conditions with end-labeled, shorter oligomer repeats (e.g., 32 personal observations). Several studies have... 

This protocol is a modification of the procedure described by Birnboim and Doly (1979). For most bacteriophage PI clones, the protocol yields a sufficient amount of DNA such that 15-20 ll can be used per restriction digest in a Southern blot. We typically use 1.5 pi per sHde for in situ hybridizations with polytene chromosomes. DNA yield strongly depends on the specific bacteriophage PI clone and may range from 3 to 15 pg. 

There have been several clinical trials of liposome/DNA complexes, although almost all of these have been involved in the treatment of cystic fibrosis. Most protocols involve the use of DC-chol/DOPE liposomes directly instilled onto the nasal epithelium ofCF patients. The effect of gene expression on CFTR function was determined and the presence of the gene in the target cells was determined by PCR (polymerase chain reaction) and Southern blot analysis. The results from these trials are described in Section 3. Initial clinical studies found no evidence of any safety problems with the use of liposome/DNA complexes. This is surprising as it is well documented that the liposome/DNA complexes used in clinical trials are directly cytotoxic in vitro. Furthermore, studies in mice and macaques have demonstrated that exposure to high doses or to repeat doses of... 

Ziegler RG, Blot WJ, Hoover R, Blattner WA and Fraumeni JF Jr. 1981. Protocol for a study of nutritional factors and the low risk of colon cancer in Southern retirement areas. Cancer Res 41 3724-3726. 

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