Stringency, of hybridization

Hybridization conditions. The stringency of hybridization conditions is such as to ensure specific hybridization between probes and standard DNA preparations and the drug substances must not interfere with hybridization at the concentrations used. 

After hybridization, process the filters in 100 mL of wash buffer at 40°C for 10-20 min. One or two washes are usually sufficient to remove background hybridization signals. See Note 7 for further discussion of stringency of hybridization and washing conditions. 

Fidelity of hybridization Does a reduction in stringency increase hybridization Does an increase in stringency reduce hybridization Does competition of hybridization with an unlabeled but identical probe reduce hybridization ... 

Because of the sequence complexity of genomic DNA, isolation of false positive clones by blot hybridization is a significant problem when the stringency of the washing conditions is very low (wash temperature <42°C). 

After Southern transfer of restriction-digested genomic DNA, filters are prehybridized for 1-4 hr. The probe is boiled for 10 min to denature it, added to fresh hybridization buffer, and hybridized overnight. To calculate appropriate hybridization temperatures in buffers with or without forma-mide, and for details of hybridization buffers and washes, see standard protocols.13 For hybridizations at low stringency to detect signals from fragments of approximately 65-70% overall sequence similarity, we apply 55° for hybridization and washes in 6X SSC without formamide. Similar protocols can be used for colony and plaque hybridizations. 

Hybridization Impinging on the rate and specificity of hybridization are the factors associated with stringency, especially temperature, salt concentration, and formamide concentration. Other factors include probe length and concentration. These parameters are empirically optimized. 

Five steps can be distinguished in membrane hybridization (i) immobilization of target nucleic acid (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe non-specifically (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes. 

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