Factors Influencing Hybridization

The kinetics of nucleic acid hybridization rely heavily on such parameters as temperature, salt concentration, solvent concentration, and the relative strength of nucleic acid hybrids (RNA : RNA > RNA : DNA > DNA : DNA) (6). Blots are typically prehybridized (at the hybridization temperature) in a solution that reduces nonspecific binding of labeled DNA during hybridization. The prehybridization solution is then replaced with hybridization solution, which contains the labeled probe. Composition of prehybridization and hybridization solutions can vary depending on the protocol and subsequent detection method. Two commonly used formulations are presented in Table 2. The temperature of hybridization largely determines the specificity of the signal obtained in Northern and Southern analysis (high temperatures increase stringency). Many protocols recommend the use of formamide-based prehybridization and hybridization solutions to lower the required hybridization temperature without compromising stringency. Hybridization can be carried out in sealed plastic bags, although commercially available hybridization bottles and ovens are recommended as a safe and reliable alternative. Some protocols recommend the use of high-molecular-mass polyethylene glycol or dextran sulfate in the hybridization solution to concentrate the probe and enhance the final signal (6,7).

12.2. WASHING AND VISUALIZATION OF NUCLEIC ACID BLOTS After hybridization the, nonspecifically bound probe is removed by sequential washes with buffer (see Tables 1 and 3). The temperature and composition of the wash buffer have an effect on the specificity of the signal obtained. Higher temperatures and/or lower salt concentrations increase the stringency of the wash and remove the probe that is not strongly hybridized to complementary sequences. When radioactive probes are utilized, the washing process can be easily monitored with a Geiger counter. Visualization of hybridized radiolabeled probes is accomplished by exposing the membrane to X-ray film. The use of tungstate-based intensifying screens can intensify a positive signal 10-fold when applied at -70°C.

12.3. REMOVAL OF BOUND PROBES FROM NUCLEIC ACID BLOTS If nitrocellulose or nylon membranes do not dry out during the blotting process, they can be stripped and reprobed. Probes can be removed from nitrocellulose by immersing the membrane in hot elution buffer (see Table 2) (6). Stripping of nylon membranes (DNA or RNA) can be accomplished using one of the following:

1. Immersing the filter in 1mM Tris-HCl, 1mM EDTA, and 0.1X Denhardt's reagent for 2 h at 75°C;

2. Immersing the membrane in a formamide-based stripping solution containing 2X SSPE and 50% formamide for 1 h at 65°C;

3. Pouring boiling water over the membrane and letting it stand for 5-10 min.

Probes can also be removed from DNA blots by alkaline treatment with sodium hydroxide at 42°C, followed by neutralization in Tris buffer at 42°C (7). Once the probe is removed, the hybridization membrane should be air-dried briefly and sealed in a plastic bag. Sealed blots can be stored at 4°C for extended periods of time without deterioration.

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