Southern nylon membrane

Transfer by blotting to a nylon membrane allows the heat treatment involved in hybridisation to take place. Southern blotting technique. 

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. 

Various formulations of nylon membrane filters have appeared on the market in recent years.32 The filter material is less brittle than nitrocellulose, and the stable linkages formed with DNA allow stripping off probes by heat while still retaining the target DNA on the filter, thus allowing reuse. The membranes have proved particularly effective for Southern blot hybridizations and are quite amenable to use in slot-blot and dot-blot devices, which allow application of up to 96 DNAs on a single filter. The membranes can be incubated in thermostable plastic pouches that require only a small volume of buffer. However, quantitation of DNA hybridizations on these filters has been no better than for nitrocellulose (C. P. Kurtzman, unpublished, 1987). 

Figure 2-14. Southern and Northern blotting using non-radioactive detection. Instead of using radioactive DNA probes to detect specific DNA or RNA sequences by hybridisation on a nitrocellulose or nylon membrane, a complementary DNA molecule coupled to a hapten is used. The hapten, e.g., digoxygenin as...

Methodologically, both techniques are based on the digestion of the genomic DNA with restriction endonucleases, separation of the fragments by electrophoresis, followed by X transfer to a nylon membrane and finally, detection by hybridization with either a radioactive or chemiluminescent probe. This technique is known as Southern blot analysis. 

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. 

Fig. 7. Southern blotting. DNA fragments that have been separated by gel electrophoresis are transferred to nylon membrane, such as Nytran, as shown in the diagram and as described in the text.

This procedure was introduced by E.M. Southern in 1975, and is used to transfer DNA from agarose gels onto a nitrocellulose or nylon membrane for subsequent detection. Transfer to nitrocellulose is accomplished after the gel has been soaked in a denaturing solution containing 1.5 M NaCl and 0.5 M NaOH this disrupts the base pairing of double-stranded DNA, so that only the single-stranded form is present in the gel. The gel is then neutralized to a pH of 8, and placed in a transfer apparatus such as the one shown in Figure 9.14. 

In 1975 Edward Southern developed a technique that is widely used to detect fragments of DNA. This technique, known as Southern blotting,first requires an electrophoretic separation of DNA or DNA fragments by AGE. Next a strip of nitrocellulose or a nylon membrane is laid over the agarose gel, and the DNAs or DNA fragments are... 

A survey on molecular biology products (Ausubel et at, 1991) indicated that for nitrocellulose about 70% of respondents preferred S S NC (other manufacturers < 10%) but that the preference for nylon membranes differed little among Schleicher Schuell, Dupont NEN and Amersham. In the same survey, respondents had no clear preference for either nylon or nitrocellulose for Southern hybridization but a clear preference for nitrocellulose in the case of colony/plaque hybridization (where concentration of target is sufficient highly but background should be suppressed). 

For transfer to diazotized membranes (Wahl et al., 1987a,b), DNA is fractionated by electrophoresis as for other Southern procedures. However, after electrophoresis, the gel is rinsed with water and soaked in 1 M NaOAc (pH 4.0, 30 min) followed by rinsing in water and soaking in 20 mM NaOAc for 30 min. Transfer to diazotized membranes is as for nitrocellulose except that 20 mM NaOAc is used as a blotting buffer. Since DNA becomes covalently linked, it is not necessary to bake the membranes. Nylon membranes are generally preferred over these membranes for hybridization. 

Nylon, particularly the positively charged membranes, has largely replaced nitrocellulose or diazotized paper for hybrid selection. DNA, e.g., from linearized plasmid, can be applied in 0.4 N NaOH to denature the DNA and to promote a strong fixation. The nylon membrane (2-cm squares) is soaked for 5 min in water and then 30 min in 0.4 N NaOH. Concurrently, DNA is denatured for 10 min in 0.4 N NaOH. DNA is then applied repeatedly with drying between applications. The membrane is then washed twice with 1 M NH4OAC and twice with 1 X SSG. The membrane is blotted and dried (may contain > 100 p,g/cm ). An alternative method is presented in Section 8.3.1. Disks of 0.5 cm are then punched out with a sterile one-hole paper punch. It is also possible to use Southern blots, or fragments thereof, for hybrid selection. 

Hybridization. Genomic DNA (c. 3 pg)of E. graminis f. sp. horde was digested with the restriction endonucleases Hind III, Sst 1, Pst 1, Xho 1, Sal l, Eco R1 and Bam HI in accordance with the manufacturer s Instructions (BRL), separated by 0.7% agarose gel electrophoresis, and then Southern blotted (45) onto nylon membrane (Hybond-N Amersham International). Prehybridization and hybridization reactions were done at 42°C using 40% deionised formamide, and 5 x SSC and 5 x Denhards solution. 

In the laboratory. DNA can be cut into small pieces by enzymes called restriction endonucleases. The.se enzymes are restricted to cutting the DNA at specific base pair sequences. Tbe presence of polymorphic sites, where there are differences in the base pair sequence of the DNA, may create or abolish a cutting site for a particular restriction endonuclease. In these instances, the polymorphisms are known as restriction fragment length polymorphisms (Rf-LPs) as they lead to the production of DNA fragments of different lengths after enzyme action. DNA fragments may be separated by electrophoresis, transfered to a nylon membrane and hybridized with a DNA probe labelled with a radioactive or optically active marker. This is called. Southern blot analysis (Fig. 1). 

With a northern blot, named for its similarity to the Southern blot, total RNA (or total mRNA) is isolated from samples and ran on an electrophoresis gel to separate sequences by their size. The RNA from the gel is then immobilized by transfer to a nylon membrane and incubated with a DNA probe labeled with The probe molecule hybridizes only to the complementary sequence, which can then be visualized on the gel by exposing an X-ray film to it. 

Two routine methods of preparing blots are employed depending upon the membrane chosen Southern transfer must be used for nitrocellulose and can be used for all nylon varieties. Alternatively, some nylon membranes are amenable to alkali transfer 1. 

The probe can be tested by binding a small amount of the labeled probe (0.5-2 pL) to a nitrocellulose or nylon membrane.The incorporation of label is then detected using an enzymic detection system as described in nonradioactive Southern hybridization detection kits (e.g., Boehringer Mannheim or see Chapter 18, Section 3.2.). 

Southern cross hybridization Southern cross hybridization was carried out by the method of Potter and Dressier (8) to construct the restriction map of the plasmid. An agarose gel with a wide sample well (11.5 x 0. 2 cm) was placed on a cooling plate of a horizontal electrophoresis apparatus. The isotope-labelled and non-labelled restriction fragments of the plasmid were separately electrophoresed and blotted on the nylon membranes. 

---