Practice question

Double digestion employs two different restriction enzymes in same reaction buffer with compatible salt and temperature conditions. If only one enzyme cuts, linear DNA yields two fragments, while failure of second enzyme leaves pattern unchanged. Appearance of two distinct bands from a linear substrate whose combined size equals parental length indicates each terminus was generated by different enzyme, meaning both enzymes successfully cleaved their respective sites. Failed digestion by one enzyme would leave single band or parental pattern. Thus two-band pattern confirms simultaneous activity of both enzymes and validates buffer compatibility and presence of both sites.