Southern Blot

The Southern blot was developed by E.M. Southern in 1975 and was the first molecular biology tool to have a major impact on clinical molecular pathology. The Southern blot is still used today, though it is being replaced by amplification methods. The implementation of Southern blot was based on prior knowledge of nucleic acid isolation, gel electrophoresis, RE digestion, and nucleic acid probe labeling for detection of DNA sequences of interest.

The Southern blot is a relatively labor-intensive, time-consuming clinical laboratory method.9 High-quality DNA is isolated from a patient specimen, subjected to RE digestion, and then fractionated by gel electrophoresis. "Blotting" is the transfer of fractionated DNA from the gel to a solid support such as a nylon membrane. The DNA is then hybridized to a small piece of complementary DNA labeled in a variety of ways and called a probe. This detection step allows the gene of interest to stand out from the vast background of DNA present in the sample. If the pattern of banding visualized on the membrane is different from the normal pattern, this may be indicative of a mutation.

Because no amplification of target DNA occurs, Southern blot analysis requires a large mass of DNA. The DNA must also be intact and of high molecular weight. Therefore, electrophoresis of the isolated DNA prior to analysis is important for assessing the integrity of the DNA, since only a small degree of DNA degradation is tolerable. Degraded DNA may produce false-negative results if a signal from high-molecular-weight DNA is expected, while false-positive results may occur if partially degraded DNA results in unusually sized bands. Fortunately, most tests in the molecular pathology laboratory today are based on PCR, which is less affected by DNA degradation. Polymorphisms within RE recognition sites also change banding patterns, a principle used to advantage in other molecular tests.

The physical movement of the DNA in the gel to the membrane may be accomplished by manual capillary transfer, automated vacuum transfer, or electrotransfer. DNA in the gel must first be "conditioned": depurination with dilute HCl and subsequent denaturation with NaOH. Dilute and brief acid treatment causes hydrolysis of the DNA phosphodiester backbone to occur spontaneously at the sites of depurination. This acid induced fragmentation facilitates efficient transfer of the highest-molecular-weight DNA species from the gel to the membrane. Alkali treatment denatures double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA), essential for subsequent nucleic acid hybridization with a labeled ssDNA probe. The

DNA is permanently fixed to the membrane by thoroughly drying the blot in an oven or by exposing the blot to a precise amount of UV irradiation.

The blot is immersed in prehybridization buffer to prepare the DNA on the blot for hybridization with a probe. Prehybridization buffers contain blocking agents included to minimize unwanted nonspecific DNA probe binding that would otherwise contribute to high background on the final image of the Southern blot used to view the results and make diagnostic conclusions. The prehybridization step equilibrates the membrane and blocks sites on the nylon membrane without DNA to prevent the probe from binding nonspecifically and increasing background. A large volume of blocking agent is therefore advantageous. Addition of the labeled probe to the blot begins the hybridization phase of the Southern blot process. A small volume of buffer is used to facilitate probe and target specifically finding each other, thereby promoting hybridization. Hybridization takes several hours to overnight at an appropriate temperature determined by multiple variables: concentrations of the two species; time permitted for hybridization; complexities of the nucleic acids involved; length of the probe and its target and their complementarity to each other (degree of mismatch); pH; temperature; and ionic strength of the buffer used.

DNA probes are labeled before use in hybridization assays to permit visualization of probe-target binding (in reverse hybridization assays, described below, unlabeled probes are immobilized and the target is labeled during the amplification step that precedes hybridization). Such labeling may be accomplished isotopically or nonisotopically. High-specific-activity DNA probes may be generated by in vitro biochemical reactions that synthesize new stretches of DNA from dNTPs, using the probe as a template. One of these dNTPs is labeled with a reporter molecule such as 32P, biotin, or digoxigenin. When incorporated into the newly synthesized DNA, the labeled dNTP, even though it is only one of the 4 dNTPs in the DNA probe, is sufficient to label the entire probe for detection. The probe is then used in vast molar excess relative to target DNA in nucleic acid hybridization to drive the hybridization reaction as quickly as possible.

After hybridization, the blot is washed with buffers containing sodium chloride and detergent to remove excess probe and reduce background. Sodium chloride concentration and stringency are inversely related: the lower the sodium chloride concentration, the more stringent the wash. Increasingly stringent washes remove more non-specifically bound probe. The temperature of the wash buffer and stringency are directly related: high-temperature washes are more stringent than lower-temperature washes and further contribute to hybridization specificity. When appropriately stringent washing of the blot is complete, only the specific hybrids of interest should remain. Visualization of these specific hybrids, which appear as bands, is achieved by autoradiography for radioactive probes or by luminography for chemiluminescent probes.

Hybridization with biotinylated probes is followed by chemical reactions, resulting in insoluble colored precipitates at the site of hybridization on the blot itself that serve as the endpoint (this is also the detection scheme used in the line probe assay; see below). Simple visual inspection is then applied for both isotopic and nonisotopic Southern blots to determine the position where the labeled probe hybridized to its target patient DNA. That position, relative to detection of appropriate controls, allows interpretation.

Northern blotting is an extension of Southern blotting that uses RNA instead of DNA as the target of investigation. Northern blotting is as labor-intensive as Southern blotting but even more problematic due to the highly labile nature of RNA. While northern blotting has been very useful in the research setting to demonstrate the selective expression of genes in various organs, tissues, or cells, it has not become a routine tool in the clinical molecular pathology laboratory.

Examples of Applications of Southern Blotting

1. B- and T-cell antigen receptor gene rearrangement for leukemia and lymphoma10

2. Fragile X syndrome diagnosis

3. Myotonic dystrophy diagnosis

0 0

Post a comment