Southern hybridization

Identifying Specific DNA Sequences by Southern Blotting (Southern Hybridization)... 

Mulbry WW, JS Karns, PC Kearney, JO Nelson, CS McDaniel, JR Wild (1986) Identification of a plasmid-borne Parathion hydrolase gene from Flavobacterium sp. by Southern hybridization with opd from Pseudomonas diminuta. Appl Environ Microbiol 51 926-930. 

Southern hybridization experiments, employing the cloned PHA synthase structural gene of Acinetobacter sp. and sucrose gradient fractions of DNA preparations separated in plasmid and chromosomal DNA fractions gave two hybridization signals and revealed some but not yet conclusive evidence for... 

PCR uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern hybridization, or direct sequence determination. 

Wood et al. (1991) have used the Southern hybridization method for detecting DNA amplification and a possible structural rearrangement of the HER-2/nen oncogene in 1 of 12 bladder tumors. Amplification of this oncogene in the tumor was sixfold that of oncogene found in placental DNA. Approximately 36% of the tumors studied overexpressed HER-2 mRNA, which was 3- to 38-fold that of normal urothelium. HER-2 overexpression occurred in superficial and invasive tumors. Deoxyribonucleic acid amplification occurs infrequently in bladder carcinoma, in contrast to its occurrence in some other carcinomas. Immunohistochemical analysis has shown that pi85 HER-2 polyclonal antibody is specific for HER-2 protein overexpression in bladder carcinoma. This study was carried out prior to the use of Herceptin. 

Figure 1-4. Southern hybridization ana DNAs with EcoRI and Eagl.

Southern Hybridization. Our hybridization procedures are essentially those of Palmer.3 New blots are washed for 1 hr at 65° in 0.1 X SSC, 0.5% (w/v) sodium dodecyl sulfate (SDS) blots that have been used previously are treated with 0.4 N NaOH at 40° for 30 min and rinsed 3 times with 2 X SSC for 10 min each time. If a large number of blots are to be hybridized simultaneously, 10 can be sealed into one bag and 50 ml of hybridization buffer added. Hybridization buffer is 4X SSC, 10 M EDTA, 5X Den-hardt s [50 X Denhardt s is 1.0% (w/v) BSA, 1.0% (w/v) Ficoll 40 (Sigma), and 1% (w/v) polyvinylpyrrolidone (PVP type 400, Sigma)], 0.5% (w/v) SDS, and 25 /tg/ml of calf thymus DNA sonicated and freshly boiled. All except the last component are Millipore (Bedford, MA) filtered before the DNA is added. For heterologous probes we prehybridize and hybridize at 55° for homologous or highly conserved DNA we use 65°. We prehybridize for a minimum of 2 hr, then replace the buffer with fresh solution. The denatured probe DNA (see below) is diluted in 5 ml buffer and added. Probe concentrations should not exceed 50 to 100 ng/ml for buffers that do... 

Southern hybridization, shown in Figure 17D, confirmed that the DNA from A. tumefaciens had been transferred into the plant... 

Talmadge, J. E. and Zbar, B. (1987). Clonality of pulmonary metastases from the bladder 6 subUne of the B16 melanoma studied by southern hybridization. J. Natl. Cancer Inst. 78, 315-320. 

F ure 1. Southern hybridization of JCM Figure 2. Southern hybridization of JCM 7664 DNA with bcsA probe 7664 DNA with bcsABUA probe... 

Lehman CM, Sarago C, Nasim S, Comerford J, Karcher DS, Garrett CT. Comparison of PCR with southern hybridization for the routine detection of immunoglobuhn heavy chain gene rearrangements. Am J Clin Pathol 1995 103 171-6. 

Beside solution assays, AMPPD and AMPGD can be used for detection of nucleic acids on membranes since the AMP D anion is hydrophobic and will attach to the membrane in some fashion in the direct vicinity upon formation. This allows its application in assays where resolution is required such as Northern and Southern hybridization. 

Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-... 

A survey on molecular biology products (Ausubel et at, 1991) indicated that for nitrocellulose about 70% of respondents preferred S S NC (other manufacturers < 10%) but that the preference for nylon membranes differed little among Schleicher Schuell, Dupont NEN and Amersham. In the same survey, respondents had no clear preference for either nylon or nitrocellulose for Southern hybridization but a clear preference for nitrocellulose in the case of colony/plaque hybridization (where concentration of target is sufficient highly but background should be suppressed). 

Add samples (in 5% glycerol with 0.05% bromophenol blue and/or xylene cyanol FF usually added as 6 x concentrate to 5 vols. of sample). Although for ordinary electrophoresis the same pipette tip can be used (rinsing in between), a separate tip is recommended for each sample for Southern hybridization. Usually 10-30 pi can be introduced per well. 

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