Hybridization Southern blot

Colleni, G.W.B., Jhanwar, S.C., Ladanyi, M. et al. (2000) Comparison of a multiplex reverse-transcriptase polymerase chain reaction for BCR-ABLto fluorescence in situ hybridization, southern blotting, and conventional cytogenetics in monitoring of patients with Phi-positive leukemias. Diag Mol Pathol, 9, 203-209. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

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

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. 

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

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 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. 

Such renaturation or annealing of complementary DNA strands is an important step in probing a Southern blot and in performing the polymerase chain reaction (reviewed in Chapter 7). In these techniques, a well-characterized probe DNA is added to a mixture of target DNA molecules. The mixed sample is denatured and then renatured, When probe DNA binds to target DNA sequences of sufScient complementarity, the process is called hybridization. 

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). 

The field of DNA microarray has evolved Ifom the key insight of Ed Southern (13), who showed that it is possible to attach nucleic acid to solid support. The resulting Southern blot can be viewed has the first DNA array (14). It was only a small step to improve the technique to filter-based screening of clone libraries, which introduced a one-to-one correspondence between the clone and the hybridization signal (15) in a fixed position in this way the clone could be uniquely identified and information about it accumulated. 

Another variant of the hybridization assay is the Northern blot. Here it is RNA, not DNA, that is separated on a slab gel and transferred to a membrane. In the original version of the method, a special chemically treated cellulose membrane was used to hold the RNA, since nitrocellulose does not normally bind RNA. However, conditions have now been found where nitrocellulose will indeed retain RNA molecules. Nylon membranes can also be used. Radiolabeled DNA probes and autoradiography are then employed as above in the Southern blot method. The method is often useful in studying how levels of RNA species in a cell vary with stages of development and differentiation. 

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. 

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