The Same Individual Of Different Organizations Mitochondrial Dna Heterogeneity Of The Study

Object ive : To establish the Denaturing High-Performance Liquid Chromatography(DHPLC) method that rapidly screening mitochondrial DNA heteroplasmy in control region between multiple tissues within the same individual at the same time,we used direct sequencing to confirm the frequency of heteroplasmy ,thus we can interpritate the results more reasonabaly by mtDNA analysis.Method: 234 samples from 18 individual were collected during autopsy, including blood, heart, liver, spleen, lung, kidney, pancreas, brain, muscle, skin, rib, nail and hair,and three control samples Y1 Y2 and Y3 were selected in our lab.We select two polymorphic segments of hypervariable region that is HV I :ntl6159-16391 and HV II: nt172-408,all samples are amplified by Ampli Taq GOLD^TMDNA polymerase and the amplificational condition is optimized.And we select the control samples to establish the optimal condition of DHPLC ,we also used all samples of first individual that show no heteroplasmy by DHPLC and direct sequencing analysis to establish the model screening heteroplasmy between tissue types .Firstly,we rapidly screen heteroplasmic samples by DHPLC, The samples that appeared to be heteroplasmic detected by DHPLC analysis were confirmed by forward and reverse sequencing by more long products and electrophoresis with ABI Prism 310 Genetic analyzer. Result:The optimal temperature of DHPLC in HV I and HVII is 59.2℃and 55℃ respectively. 11 heteroplasmic individuals were screened by DHPLC,but 9 heteroplasmic individuals were confirmed by forward and reverse sequencing.There are 4 point heteropalsmy and 6 length heteroplasmy.Heteroplasmy at two sites were occurred in two of nine individuals,thereinto one individual appeared point and length heteroplasmy .It is more interesting to have all samples per individual of 4 individuals showed the same heteroplasmy at the same position. The heteroplasmy frequency of the selected 13 tissue are deference, range from 27.8% to 44.4%.Among these tissues, the frequency of heteroplasmy is highest in muscle tissue. The frequency of heteroplasmy at ntl6184-16193 and nt303-309 is higher than other heteroplasmic positions.Conclusion: DHPLC method can rapidly screen mtDNA heteroplasmy and it is economical sensitive and automatic .With the development of screening and sequencing technology,mtDNA heteroplasmy has been found more and more high level.So it was been taken into account for the interpritation of results in forensic examination by mtDNA.In forensic community we must extend the test regions of mtDNA to increase the discrimination power.