la jolla calif the ecl 3 u2032 oligolabelling and detection system amersham arlington heights ill was used for synthesis of labeled oligonucleotide probes hybridization and signal detection the oligonucleotide sequences used to detect each point mutation were substituted bases are shown as upper case letters for signal detection each oligonucleotide was labeled with fluorescein 11 dutp using terminal deoxynucleotidyl transferase according to the manufacturer 39 s instructions amersham ltd arlington heights ill the membranes were prehybridized in 5 u00d7 ssc 0 1 hybridization buffer component 0 02 sds 5 liquidblock at 42 u00b0 c for approximately 2 hours labelled oligonucleotide probes were added to individual bags containing the membranes and prehybridization buffer and incubated at 420 u00b0 c overnight the blots were washed twice with 5 u00d7 ssc 0 1 sds for 5 minutes at room temperature stringency washes for hybridization with oligonucleotides having the sequence of seq id no 30 or 28 were performed twice in 0 2 u00d7 ssc 0 1 sds for 15 minutes at 42 u00b0 c membranes probed with an oligonucleotide having the sequence of seq id no 29 was washed twice under less stringent conditions 0 5 u00d7 ssc 0 1 sds 15 minutes at 42 u00b0 c detection of a fluorescent signal was performed according to standard methods the mean age of the twenty probands was 44 u00b1 11 years range 27 62 years thirteen 65 0 were men and seven 35 0 were women eleven had iron overload one had hepatic cirrhosis two had diabetes mellitus four had arthropathy and.