Southern blot analysis

Northern and Southern blot analyses are carried out as shown in Figure 2.7 and as described below. Restriction enzyme digested DNA (Southern) or total RNA or poly(A) mRNA (Northern) is subjected to agarose gel electrophoresis, which separates DNA and RNA molecules based on their size. The separated molecules are then transferred to a nylon or nitrocellulose membrane by capillary action transfer... 

Southern blotting can be used to follow the inheritance of selected genes. Mutations within restriction sites change the sizes of restriction Ifagments and hence the positions of bands in Southern-blot analyses. The existence of genetic diversity in a population is termed polymorphism. The detected mutation may itself cause disease or it may be closely linked to one that does. Genetic diseases such as sickle-cell anemia, cystic fibrosis, and Huntington chorea can be detected by RFLP analyses. 

Southern blotting can be used to follow the inlieritance of selected genes. Mutations within restriclioii sites change the sizes of restriction fragments and her ice the positions of bands in Southern-blot analyses. 

Figure 1. (A) Southern blot analyses of Erff-digested C. reinhardtii DNA probed with, respectively, hydAl-or /lytM2-specific DNA probes. The arrows indicate the location of the restriction digest fragments. (B) Results from Northern blot analyses of RNA isolated from cultmes grown photoheterotrophically and then anaerobically-induced for different periods of time and probed with hydAl- or hydA2-specific probes. The bars represent H2-production activity measured concomitantly. Gels at the bottom show the original Northern blot data.

Reversibility of Reduction of Intracellular Extrachromosomal HBV DNA Additional experiments were then performed to assess the reversibility of ddC-inhibited expression of intracellular extrachromosomal HBV DNA. As shown by the Southern blot analyses presented in Figure 2, synthesis of intracellular extrachromosomal HBV DNA was completely inhibited after a six day treatment with ddC at all concentrations tested, including the lowest concentration, 100 ftM (Lanes 14-22). The expression of intracellular extrachromosomal HBV DNA, compared to DMSO-treated cells (Lanes 11-13, arrows), recovered after removal of ddC (Lanes 25-33, arrows). DNA obtained from HepG2 cells (Lane 8) presented no band corresponding to HBV DNA. 

TABLE 2. Data from Southern blot analyses showing the presence of specific restriction sites in the hybrid mutants X3M and X3S. Refer to Fig. 2 for the comparative site locations in spinach and Svnechocvstis. 

When selecting for loss or gain of HPRT function, the cells should be maintained in the positive or negative selection prior to the selection switch that will assay the altered HPRT activity. The transiently expressed Cre-mediated excision is often mosaic. Therefore, a PCR screen should be carefully designed to detect such situations. Additionally, Southern blot analyses should be performed as a final check on candidate clones identified by PCR. 

Fig. 2A-D. Dot-blot and Southern blot analyses of immunofractionated chromatin. The DNA extracted from immunofractionated chromatin was subjected to electrophoresis on 1.5% agarose and blotted to nitrocellulose by the method of Southern or spotted directly to nitrocellulose. The membranes were probed for active (A, B) and inactive gene (C, D) sequences. Unfractionated total T), unbound (U), and bound B) antibody column fractions are shown for DNA derived from chromatin digested for 2.5 (A, C) and 20 min (B, D) with micrococcal nuclease. (Taken from [10])...

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