All family members and controls were screened for the proposed
disease-causing mutation through an amplification refractory mutation
system (ARMS) assay.
23 To ensure
CRYBB2 specificity, 25 ng of genomic DNA was digested with 2 units
DdeI in 5 μl total volume, as previously described. The
mutation-specific ARMS primer,
5′-CTGCAGGTGGGTTGG
TTAC
T-3′, was specific to the
C-to-T transition at base pair 14 of exon 6 and, to increase
specificity, had an internal nucleotide (in bold) that did not match
either the normal or mutant allele. This primer was paired with the
exon 6 reverse PCR primer. Primers for the short tandem repeat
polymorphism marker D1S1663 (409–425 bp) were included in each
reaction as a positive control for PCR. After
DdeI
digestion, the whole mixture was incorporated into a 20-μl PCR
reaction containing PCR buffer (GeneAmp II), 5% DMSO, 200 μM of each
dNTP, 50 ng of each primer, and 1 unit of DNA polymerase
(Ampli
Taq). Reactions were performed on a thermocycler
(PTC-100; MJ Research; Watertown, MA) with an initial denaturation of 2
minutes at 94°C, followed by 36 cycles of 30 seconds at 94°C, 30
seconds at 61°C, 35 seconds at 72°C, and a final 8-minute extension
at 72°C. Products were electrophoresed on 1% agarose gel stained
with ethidium bromide and then visualized by UV transillumination.
Affected individuals had a 400-bp control band and a 220-bp
mutation-specific band, whereas unaffected individuals had only the
400-bp internal control band.