Five enzymes were used to confirm the mutations
identified
. HinfI, with recognition site 5′…
G
ANTC… 3′, was used to study the
homozygous T1012C and C906T in families A and E, respectively.
ApaI, with recognition site 5′…
GGGCC
C… 3′, and
BstNI, with
recognition site 5′… CC
(A/T)GG… 3′,
also were used to confirm the heterozygous changes C783T and T1291G in
families B and C, respectively. Similarly,
DrdI, with
recognition site 5′… GACNNNN
NNGTC…
3′, was used to confirm the homozygous C1309T mutation in family D. And
finally,
BanII, with recognition site 5′… G (A/G) GC
(T/C)
C… 3′, was used to confirm the
homozygous G905T change in family E.
The PCR products digested by HinfI, BstN I, and BanII restriction enzymes were analyzed by 6% nondenaturing
polyacrylamide gel electrophoresis (Protogel; National Diagnostics,
Atlanta, GA) and were visualized by staining with ethidium bromide.
Products digested by ApaI and Drd I were analyzed
on 3% agarose gels (Bio-Rad, Herts, UK).