Sequence of normal and mutant alleles of vWF DNA and PCR analysis of patient DNA. (A and B) PCR products containing vWF exon 28 sequence were amplified from genomic DNA and subcloned as described in Materials and Methods. Representative sequences of clones containing the normal allele and mutant allele for Family A (A) and Family B (B) are shown in the region of the point mutation in the mutant allele. As indicated in the translated sequence below, the missense point mutation in nt 4105T → A results in a Phe606Ile amino acid substitution for Family A (A) and the missense point mutation in nt 4273A → T results in a Ile662Phe amino acid substitution for Family B (B). (C) In Family A, a restriction site for Bcl I was introduced only in the mutant allele by PCR using specially-constructed primers (Vs4067 and Va4398) that flank the point mutation. The PCR products were amplified from genomic DNA from a normal control (N) or Family A patients (AIII.1 and AII.1). When the PCR product (336 bp) is digested with Bcl I, only the mutant vWF allele is cut into two fragments, 302 and 34 bp long. (D) In Family B, a native restriction site for BstYI is lost in the mutant allele. Genomic DNA from a normal control (N) or Family B patients (BII.1, BIII.1, and BIII.2) is amplified by PCR with primers VsI27-3 and Va5040-5020 followed by a second round of PCR with primers Vs3673:Nsi I and Va4488-4462:Nco I. When PCR product (815 bp) is digested withBstYI, only the normal allele is cut into two fragments, 598 and 217 bp long. Molecular weight standards (bp) are shown on the side.