Nucleic acid hybridization stringency

In this phase, hydrogen bonds are created between the bases of the probe and the complementary sequences inside targeted nucleic acids. The stringency of the hybridization depends on many conditions such as the temperature of the hybridization. The hybridization is usually performed at 37 °C for 1-3 h or overnight, and increasing the temperature enhances the stringency. A low salt concentration and a high formamide concentration in the hybridization buffer also increase the specificity of hybridization. 

Despite the decrease in annealing rate, formamide presents unique advantages in nucleic acid hybridization. It is used to lower the hybridization temperature and to reduce the risk of thermal strand scissions or to control the stringency of annealing (36). A 30—50% formamide concentration allows optimal hybridization to be performed at 30—42°C rather than at 68°C without formamide. 

The concentration of a specific nucleic acid sequence in a sample can be measured by hybridization with a suitable labeled DNA probe. After hybridization, nuclease is used to destroy unhybridized probe and the probe remaining is a measure of the concentration of the target sequence. The hybridization conditions can be altered to ensure that only identical sequences (high stringency conditions) or identical plus related sequences (low stringency conditions) will hybridize with the probe and hence be detected. 

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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