DNA‐DNA Hybridization on Nitrocellulose Filters

The kinetics of DNA‐DNA filter hybridization have been studied using model systems. The results suggest that two limiting reactions occur. a) When the hybridization reaction is fast ( i.e. with a DNA of low complexity such as phage T7 at ⋝ 5 μg DNA per filter) the reaction becomes controlled by the diffusion of DNA to the filter. This is shown by the increase in the reaction rate by shaking the hybridization vials, the inverse dependence of the rate on the molecular weight of the DNA in solution and its independence on the kinetic complexity of the DNA. b) When the hybridization reaction is slow ( i.e. with a DNA of high complexity or, for T7 DNA ⋜ 2.5 μg DNA per filter disc) the reaction is limited by the hybridization reaction itself. This is shown by the inverse relationship of the hybridization rate to the complexity of the DNA and the direct proportionality of initial rate to concentration. The reaction rate is independent of shaking. 2. The hybridization‐limited reaction shows two anomalies. The reaction is independent of the molecular weight of the DNA instead of the expected dependence on the square root of the molecular weight, and the calculated rate constant is nearly 10 times too low. We suggest that these anomalies result from inadequate accessibility of the DNA fixed on the filter. 3. Our results provide practical guide lines to minimize competing renaturation reactions in filter hybridization experiments.