NEBCutter software (New England Biolabs) was used to determine
various restriction sites in normal and IVS9 + 1G N A mutant alleles.
The enzyme HphI was determined to cleave in 2 positions on the normal
allele, but 3 on the mutant allele as mutation IVS9+1GNA creates an
additional HphI cut sites. After digestion with HphI, samples were run
in an 8% acrylamide gel and visualized with ethidium bromide. The normal
control has fragment lengths of only 145, 74 and 33 bp (Fig 3, lane
4). The fragment lengths for patient 1 were revealed to be 145, 107, 74,
38 and 33 bp, which is consistent with IVS9+1GNA in the heterozygous
form (Fig 3, lane 3)