Medical Genetics - V. M. Zaporozhan 2005

Methods of Diagnosing Hereditary Diseases
Molecular-Genetic Methods (DNA Diagnostic Methods)
Other DNA Diagnostic Methods — Southern Blot Hybridization

The method was proposed in 1975 by the English researcher E. M. Southern. When total human genomic DNA is treated with restriction Enzymes, a vast number of fragments of varying lengths is generated. Standard Electrophoresis fails to identify individual DNA fragments; instead, restriction-digested genomic DNA produces a uniform smear across the entire length of the gel. Identifying specific DNA fragments in such a gel is only possible by Hybridization with labeled DNA probes. This is achieved using Southern blot hybridization (Southern blotting).

The Procedure consists of the following steps:

1) DNA Isolation and Purification. High-purity DNA is essential. To isolate the DNA, the biological material is treated with Proteolytic Enzymes to remove all Proteins. The DNA is then adsorbed onto porous Supports or extracted with organic Solvents, followed by multiple purification steps. This yields the total cellular DNA (genomic DNA);

2) DNA Digestion with restriction enzymes. The genomic DNA is treated with a restriction endonuclease. Afterwards, electrophoresis of the resulting DNA Restriction fragments is carried out in an agarose gel. However, identifying fragments directly within the gel is difficult in this case, as the gel contains a continuous smear of fragments of various lengths;

3) Blotting. The next step of the method is blotting—The transfer of fragments from the gel to a nitrocellulose or nylon filter (Fig. 10.11). The gel is first treated with alkalis, which leads to DNA Denaturation (breaking the Hydrogen Bonds between complementary NUCLEOTIDES in the two DNA strands). The gel is then placed in a buffer solution, a nitrocellulose filter is placed on top of it, followed by a stack of filter paper. The paper absorbs the saline solution, which rises upward and draws the denatured DNA fragments onto the filter. The filter has a pore size specifically chosen to prevent DNA from passing through it, capturing an exact replica of the restriction fragment distribution from the gel;

4) Hybridization with DNA probes. The DNA fixed on the filter is hybridized with DNA probes. A probe is a single-stranded DNA complementary to a specific sequence of the Gene under study. Artificially synthesized oligonucleotide DNA sequences no longer than 30 nucleotides are frequently used as probes, mostly derived from frequently occurring non-coding sequences. Probes can be labeled radioactively or with chemical agents, such as fluorescent Dyes. Radioactively labeled probes synthesized from nucleotides containing the radioactive isotope ³²P provide very high sensitivity, but they are short-lived and hazardous. Today, priority is given to chemically labeled probes;

5) Analysis of the results. The filter is washed to remove excess probe. If radioactively labeled probes are used, an X-ray film is applied to the filter. Exposure occurs on the film wherever the probe has hybridized with the DNA. THE POSITION OF the target DNA fragment can then be determined using autoradiography (Fig. 10.12).

If chemical labels are used, hybridization results can be visualized under visible or ultraviolet light (depending on the type of dye used).

Thus, blot hybridization makes it possible to identify specific nucleotide sequences within a total pool of genomic DNA restriction fragments. When a mutation affects the recognition site of the restriction enzyme used in Southern blotting, the absence of the corresponding band or the appearance of an atypical DNA fragment can be clearly recorded on the film. Elongation or shortening of a DNA fragment results in a band appearing at an atypical position.

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Fig. 10.11. Transfer of DNA from an agarose gel to a membrane filter using the Southern blotting method

There are several modifications of Southern blotting that bypass prior restriction and electrophoresis. Genomic DNA is applied directly to the filter and hybridized with probes.

While Southern blot hybridization has high sensitivity, it is quite laborious, imposes strict requirements on DNA purification, and requires a large amount of genomic DNA. Furthermore, using radioactive probes necessitates specially equipped laboratories. The time and labor intensity of the entire procedure make this method quite expensive.

Today, most laboratories use the Polymerase Chain Reaction (PCR), which is cheaper and no less accurate.

Nevertheless, Southern blot hybridization is still used for the Diagnosis of Monogenic Disorders—specifically when PCR cannot be applied for various reasons. For instance, PCR requires precise knowledge of the coding regions of a gene and their flanking intron sequences (without which primers to initiate the reaction cannot be synthesized). Certain gene regions containing exceptionally long tandem trinucleotide repeats or more complex repeats cannot be efficiently amplified. In addition, difficulties arise in amplifying large and complex genes (such as the dystrophin gene), detecting extensive deletions in a heterozygous state, and in several other cases.