Genomic Southern blot hybridization

Southern blot hybridization analysis proved that homologes of acyA and acyBl were almost perfectly conserved in the genomes of streptomycetes producing the... 

It s also possible to select your DNA before you put it in the vector. If you know the sequence (or even part of it), DNA pieces (from genomic DNA or cDNA) with this sequence can be purified on a gel and identified by hybridization to an oligonucleotide using a Southern blot. Alternatively, if you know the sequence of the ends of your DNA, you can amplify it specifically by the polymerase chain reaction. There are lots of clever ways to find your DNA. 

The localization of bNOS to the human genome was accomplished by Kishimoto and coworkers70. These investigators used a rat cerebellar cDNA to obtain a human cDNA from Clontech. This cDNA was hybridized to Southern blots containing DNA from a battery of human-rodent somatic cell DNA. Since the blots had been shown to be selective, the authors showed that the cDNA hybridized to chromosome 12. By using restriction nucleases EcoRI and Hind in the assignment was made to 12 ql4-qter. One or two copies were indicated in vivo but reducing the hybrid conditions showed more bands. It is necessary to conduct further studies to see whether the other cDNA are derived from this or another clone. 

Southern blot analysis is used to confirm the PCR results. The genomic DNA is isolated from PCR-positive ES cell clones. With the choice of restriction digest and probes for hybridization, the wild-type allele can readily be distinguished from the targeted allele since predicted novel restriction fragments are generated by the homologous recombination event. 

All. Anagnostopoulos, I., Herbst, H., Niedobitek, G., and Stein, H., Demonstration of monoclonal EBV genomes in Hodgkin s disease and Ki-1-positive anaplastic large cell lymphoma by combined Southern blot and in situ hybridization. Blood 74, 810-816 (1989). 

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