The result of the polymerase chain reaction was tested by agarose gel
electrophoresis of a 4 
l sample. The positive samples were ethanol
precipitated (see section EtOH) and resuspended in 10 to 20 l of
water for one to two restriction digests, respectively. The total digest
reaction volume was 20 l, enzyme buffer (tenfold) and BSA were added
following the manufacturers recommendations.
The digests for differentiation of the point mutation at position 1309 and 1515 were both incubated at 37psy176 C with 5 units of BfaI (New England Biolabs) added. The digest for the polymorphism at position 1786 was incubated at 37psy176 C with 6 units of KpnI (AGS) added, whereas the digest for differentiation of the polymorphism at position 2316 was incubated at 65psy176 C with 8 units of BsrDI (New England Biolabs). All assays were incubated for 5 to 10 hours.