DNA Southern blot

In situ hybridization was derived from the techniques of molecular hybridization of nucleic acids that are isolated from a particular cell population or tissue and bound to solid supports. Hybridization of such averaged membrane-bound nucleic acids identifies different classes of DNA (Southern blot) and RNA (Northern blot). However, ISH fills the gap between the detection of a specific sequence and its precise location within the tissue or the cell (Chevalier et al., 1997). 

Techniques based solely on hybridization circumvent the use of DNA polymerase enzyme. Instead, the DNA is labeled (radioactivity, fluorescence or luminescence) and a distinct signal is detectable when successful hybridization of complementary sequences has occurred. These techniques were in the past mainly used to detect longer sequence stretches in DNA (Southern blotting), but were never used to analyze single bases in a sequence. Since miniaturization, immobihzation, automation, iimovative dyes, and laser technology have revolutionized technical opportunities, it is in some cases possible... 

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...

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

The northern blotting technique is similar to the Southern blotting technique with the exception that northern blots detect specific sequences of RNA, not DNA. 

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

A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure. 

If the genetic lesion is understood and a specific probe is available, prenatal diagnosis is possible. DNA from cells collected from as little as 10 mL of amniotic fluid (or by chorionic villus biopsy) can be analyzed by Southern blot transfer. A fetus with the restriction pattern AA in Figure 40-10 does not have sickle cell disease, nor is it a carrier. A fetus with the SS pattern will develop the disease. Probes are now available for this type of analysis of many genetic diseases. 

Southern blot A method for transferring DNA from an agarose gel to nitrocellulose filter, on which the DNA can be detected by a suitable probe (eg, complementary DNA or RNA). 

The XE6 DNA was digested with several restriction enzymes and analysed via Southern blots using a probe derived fi-om the pgaW gene. A restriction map of the X.E6 clone revealed that the complete gene should be present on a 3.0 kb EcoRl fi agment (see Fig. 1). 

Siles-Lucas, M., Felleisen, R., Cuesta-Bandera, C., Gottstein, B. and Eckert, J. (1994) Comparative genetic analysis of Swiss and Spanish isolates of Echinococcus granulosus by Southern blot hybridisation and random amplified polymorphic DNA technique. Applied Parasitology 35, 107-117. 

Southern blotting Technique used to identify and locate DNA sequences which are complementary to another piece of DNA called probe using electrophorectic gels for separation of DNA and membrane filters with radiolabelled complementary probes. 

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