Restriction digest with Mnl I. The mutation identified in Prothrombin Greenville predicts the loss of anMnl I restriction site where the sequence GAGG in the normal is the enzyme recognition site. This site is converted to AAGG in the mutant. Exon 13 for the proband and five family members was amplified by PCR and subjected to digestion with Mnl I. Hydrolysis was continued for 7 hours at 37°C. Additional enzyme was added to samples for lanes 3, 4, and 6 after 6 hours of hydrolysis to ensure complete hydrolysis. Shown are the final hydrolysis products after agarose gel electrophoresis and ethidium bromide staining. Lane numbers correspond to samples obtained from individuals shown in the pedigree (Fig 5). Shown in the lane at left is a 100-base pair ladder.