Restriction enzyme digest analysis for the 5312—19A>C and 4751A>G sequence variations from family 17. (A) The VWF DNA from exons 29-31 was PCR amplified with primers 29F and 31R. Bsp1286I cuts the wild-type PCR product once, yielding bands of 893 bp and 131 bp, as is observed in the unaffected members in lanes 4 and 5. The 5312A>C sequence variation creates an additional Bsp1286I restriction enzyme site cutting the 893-bp band once, yielding 2 fragments of 644 bp and 249 bp. Four bands of 893 bp, 644 bp, 249 bp, and 131 bp are observed in the members heterozygous for this change (lanes 2, 3). (B) The 4751A>C mutation in exon 28 (amplified with primers 28CF and 28DR) destroys a restriction enzyme site for KpnI. Two bands of 389 bp and 380 bp were observed in the unaffected members (lanes 4, 5), and 3 bands of 389 bp, 380 bp (cut), and 769 bp (uncut) were observed in the affected members who are heterozygous for this change (lanes 2, 3). Lane 1 is a 100-bp ladder.