126 Pingoud, Alves, and GeigerTable 4
Resolution of Different Gel Systems for Electrophoresis of DNA Fragments
Separation range of linear DNA in bp Appropriate gel system
5-10 20%PAA
10-50 15%PAA
50-100 10% PAA
100-200 5% PAA
200-1000 3% PAA
100-500 3% Agarose
200-3000 2% Agarose
500-6000 1% Agarose
1000-20,000 0.5% Agarose
5000-60,000 0.3% Agarose
50,000-several million 1% Agarose (FIGE)specific activity on substrate concentration was observed: It was dem-
onstrated that cleavage of refractory DNA recognition sites could be
achieved by addition of DNA that contains an abundance of EcoRII-
sensitive sites. It was argued that EcoRII may be the prototype restric-
tion endonuclease that requires at least two simultaneously bound
substrates for its activation (177). A similar observation was recently
made for NaeI (178). It was shown that some NaeI sites are cleaved
rapidly and that some are almost totally resistant to cleavage. NaeI could
be activated to cleave these resistant sites in the presence of excess cleav-
able NaeI sites. Interestingly, with spermidine present, resistant sites
were cleaved rapidly and cleavable DNA inhibited their cleavage.
3.3. Assay for Restriction Endonuclease Activity
The earliest attempts to determine the activity of a restriction enzyme
made use of the drop in viscosity of DNA solutions on nucleolytic
degradation of the DNA (5). This rather insensitive and imprecise
method was subsequently replaced by the analysis of the reaction
products obtained from restriction enzyme catalyzed digests of DNA
by gel electrophoresis in which fragments are separated according to
size (179) (for the separation properties of different electrophoresis
gels, see Table 4). DNA bands are usually visualized by staining the
gel, preferentially after electrophoresis, in a solution of ethidium
bromide, which binds to DNA by intercalation and thereby increases
its fluorescence quantum yield about 20-fold. As little as nanogram