Southern hybridization technique

For both scenarios the polymerase chain reaction (PCR) can be used. Alternatively, in the second case, a hybridization technique such as Southern blot (see later) can be applied. 

Southern [DNA] (32) blots). Recently, a technique has been described for treating nucleic acid target-probe complexes as antigens in an antigen capture system performed in plastic ELISA plates (38). The latter technique is of interest because it allows automated equipment designed for performing standard ELISA to be used for nucleic acid hybridization techniques. 

Due to the small volumes and feature sizes, reaction rates are found to be quite different in the microbiosensor system in comparison to their macro counter part. Most of this is due to the fact that diffusion is not the limiting factor in a reaction any longer. For example, the diffusion time of a particle with a diffusion coefficient = 10 m s is 15 min to travel a distance of 1 mm, but only 10 s to travel 100 /rm and only 0.1 s to travel 10/rm [37]. Therefore, DNA hybridization reactions, antibody-antigen binding events, and enzyme—substrate catalytic reactions take place in a fraction of the time required earlier. DNA hybridization can be accomplished in a matter of seconds in a microchannel system, while it takes in the order of an hour when employing standard Northern or Southern Blotting techniques with a piece of nylon membrane soaking in several milliliters of hybridization solution. 

Biotechnology is being used in the diagnosis of disease. For example, samples of DNA are treated with restriction endonucleases and then subjected to electrophoresis on gels. Using the Southern blot technique, the x restriction fragments on the gel are hybridized with radioactive cDNA... 

Northern blot technique A technique that is used to identify RNA. RNA is separated according to size by use of a denaturing gel and electrophoresis prior to being blotted onto a solid support. The mRNA transcripts are then detected by hybridization with a radioactive labelled probe. The abundance of the mRNA is indicated by the intensity of the radioactive signal. See also Southern blot technique. NOS nitric oxide synthase, nosocomial synonomous with HAL NO synthase See nitric oxide synthase. 

Cellular DNA is extracted, resuspended, restriction enzyme digested, and elec-trophoresed in a 1% agarose gel, and transferred via Southern blot technique to nitrocellulose membranes. The nitrocellulose is baked, prehybridized, hybridized, washed, quantitatively analyzed for radioactivity, and exposed to film. 

It is necessary to prepare undegraded DNA to take full advantage of pulsed-field gel electrophoresis. This can usually not be achieved by the standard methods presented in Chapter 3. Techniques for the preparation of very high molecular weight DNA are given in Table 9.4. After electrophoresis of this DNA (Table 9.5), the gel is stained by immersion in a solution containing 0.5 p-g/ml of ethidium bromide for about 60 min and destaining in water for about 1 day. Southern hybridization can then be performed after the exposure to acid and base to achieve partial hydrolysis and complete denaturation prior to transfer. Possible problems and remedies are summarized in Table 9.6. 

Southern s technique could not be applied to the blot transfer of RNA separated by gel electrophoresis. Alwine et al. (A2) therefore devised a procedure in which RNA bands are blot transferred from the gel onto the chemically reactive paper, where they are bound covalently. The reactive paper is prepared by diazotization of aminobenzyl oxymethyl paper, which can be prepared from Whatman paper by a series of uncomplicated reactions. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes. Hybridizing bands are detected autoradiographically. This method is termed Northern blotting. 

DNA arrays, like more traditional hybridization techniques such as Southern and Northern blotting, make use of the fact that single-stranded nucleic acid species (DNA and RNA) that possess sequences complementary to each other will hybridize together with exquisite specificity to form double-stranded complexes. In the traditional approaches, the samples to be analyzed are distributed according to size by gel electrophoresis, transferred to and immobilized on solid membrane supports, and probed with specific, labeled complementary nucleic acid sequences. 

DNA Techniques for the Differentiation of Animai Species Southern Hybridization... 

The oldest DNA technique in food analysis is based on labeled probes, which bind directly to DNA if complementary regions exist. This method, called Southern hybridization, is very time consuming. Further disadvantages are insufficient sensitivity for many questions, as well as the problem of nonspecific hybridization signals for closely related animal species (Behrens and Unthan, 1999). 

The principle behind DNA chips was bom from the basic Southern blot technique, and it is now a fast-moving field at the cutting edge of genome research. The two main scientific elements of DNA chips are the manufacture of microarrays with single-stranded oligonucleotides attached to pixels on the surface and hybridization analysis with spatial resolution. 

Protocol 29.1 Preparation of HMW DNA from Embryos, 526 RESTRICTION DIGESTION OF HMW DNA, 531 Protocol 29.2 Restriction Digestion of HMW DNA, 531 PUESED-FIEED GEE EEECTROPHORESIS, 533 Protocol 29.3 PFGE Technique, 534 SOUTHERN HYBRIDIZATION ANAEYSIS, 536 Protocol 29.4 Transfer of DNA to a Membrane, 536 Probe Preparation and Hybridization, 537 Data Analysis, 537... 

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. 

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