SLC25A13 Gene Mutation Analyzed By PCR-reverse Dot-blotting Hybridization Method

ObjectiveTo establish a simple, rapid, sensitive and specific method to detect typeⅠ,typeⅢand typeΧmutations of SLC25A13 gene.Methods1. Design and synthesis probes.2. PCR fragment of SLC25A13 gene contain typeⅠ,typeⅢand typeΧmutations.3. Products of target DNA fragments were hybridized on nylon membrane with different probe concentration, hybridization temperature and washing membrane temperature.4. Analysis of typeⅠ,typeⅢand typeΧmutations of SLC25A13 gene with reverse dot blot hybridization method in 100 known and unknown mutation patients respectively.Results1. The more ideal results could be obtained at the following experimental conditions: 0.45μm nylon membrane with specific probes was used; the concentration of probe was 15-20pmol/μL, the temperature and time of hybridization and washing membrane were at 42.5℃for 4 hours and at 42.5℃for 5 minutes respectively.2. In known mutation group patients, 17 were homozygous typeⅠmutation, 1 was typeⅠ/ typeⅢcompound heterozygotes, 1 typeⅢ/typeⅩ, 2 typeⅠ/typeⅩ, 44 were typeⅠmutation on only one allele, 1 typeⅢmutation on one allele and 1 typeⅩon one allele, which were detected by PCR- DNA sequencing, were also found by the PCR-reverse dot blot hybridization method.3. In unknown mutation group patients, 8 homozygous typeⅠmutation, 7 typeⅠmutation on one allele, 1 typeⅢmutation on one allele and 1 typeⅩon one allele were detected by the PCR-reverse dot blot hybridization method, which were confirmed by PCR- DNA sequencing. ConclusionsTypeⅠ, typeⅢand typeⅩmutations of SLC25A13 gene can be identified simply and rapidly by .PCR-reverse dot-blot hybridization method. Only one day.