Forum pracy nad projektem wskrzeszenia The Forge

[xp052505]

China Baoan mitochondrial DNA D


Chinese papers League finishing. Key words sequence polymorphism Polymorphism of mitochondrial DNA Dloop region in Chinese BAOAN ethnic group 【Abstract】 AIM: To investigate the mitochondrial DNA sequence polymorphism in Chinese BAOAN ethnic group and to provide basic data for ethnic origin investigation and forensic purpose. METHODS: Genomic DNA was extracted from the whole blood of 98 unrelated individuals of Chinese BAOAN ethnic group by standard chelex100 method. The sequence polymorphism was determined by PCR amplification and direct sequencing. RESULTS: Sixtysix polymorphic sites and 62 haplotypes were identified in mtDNA Dloop region (np1609116418). The genetic diversity was calculated to be 0.9862, and the genetic identity was 0.0237. CONCLUSION: There are some particular polymorphism sites in Chinese BAOAN ethnic group , and these sites provide an important basis to investigation of the origin of BAOAN and the relationship between BAOAN and other ethnic groups. The result also suggests that sequence polymorphism (np1609116418) in human mitochondrial DNA can be an useful tool for forensic identity. 【Keywords】 mitochondrial DNA; sequence polymorphism; Dloop region Abstract Objective: The object of genetic groups, the group observed the genetic characteristics of mitochondrial DNA for the study of the origin of Ethnic Minorities to provide scientific basis, and provide for the forensic identification based on mitochondrial DNA sequence polymorphism data. Methods: Using PCR direct sequencing of amplified products, unrelated individuals of 98 cases of mitochondrial DNA D Baoan Central District, HVS Ⅰ sequencing. Results: The high mtDNA variable region Ⅰ (HVS Ⅰ) 16091 ~ 16418 with the Anderson sequence comparison between the total mutation found in 66, 62 haplotypes. The main form of mutation base substitution, which converted 87%, 11% transversions, insert the 1% base, base deletion of 0.5%. Baoan District, mitochondrial DNA HVS Ⅰ degree of genetic difference 0.9862, probability 0.0237 coupling. Conclusion: Bonan compared with other groups has its own unique genetic characteristics of mitochondrial DNA sequences, and Asia other ethnic groups and Caucasians were significantly different. Bonan mitochondrial DNA sequence polymorphism not only different from the western species, but also different from other East Asian ethnic origin. mitochondrial DNA control region sequences of HVS Ⅰ polymorphism can be used for forensic identification and paternity. Key words Mitochondrial DNA; sequence polymorphism; D loop region 0 Introduction mitochondrial DNA (mtDNA) is a closed circular, there are 16,569 base pairs of double-stranded molecule [1]. The non-coding region (also called control area, Dloop District) is located between tRNAPro and the tRNAPhe gene, about 1100 bases, including two highly variable Area (HVS Ⅰ and HVS Ⅱ). In the non-coding regions , especially the two hypervariable region, mtDNA exists a high degree of polymorphism, while each cell has 103 to 104 copies of mtDNA has been widely used in forensic individual identification and pro-the right identification, especially for corruption and degradation of specimens and the hair ,[link widoczny dla zalogowanych], bones and other biological samples for testing has a unique role. Minorities in Northwest China's natural environment and religious beliefs due to the restrictions, the tribes maintained a relative independence between. but many ethnic minorities do not own this text, it is their national origin of the text has been no reliable evidence. We have a unique ethnic minority groups in China as the research object, observed mtDNA population genetic characteristics of the study provide genetic ethnic origin Evidence is also provided for the forensic identification of the basis of mtDNA sequence polymorphism data. 1 objects and methods 1.1 Object 98 cases samples were collected from Baoan Linxia state product Rock Hill County, to provide blood samples of unrelated individuals each of both healthy and back 3 generations or more, to ensure that its national representation. 1.2 Methods 1.2.1DNA extract plus 5 μL The EDTA anticoagulated blood was added with 1 mL deionized water in 1.5 mL centrifuge tubes , set the bed shake shake 20 min, centrifuged 2 min, supernatant; deposits added 5 mol · L-1 solution of 200 μL of chelex oscillation 10 s, 56 ℃ water bath 20 min, 100 ℃ dry heat 8 min, oscillation 10 min , centrifuged 2 min, 4 ℃ save backup. Table 198 cases of unrelated individuals mtDNA HVS Ⅰ Baoan District base mutation and haplotype (slightly) 1.2.2mtDNA standard amplification and purification of mtDNA sequence according to design primers are as follows: H5'CAT GGG GAA GCA GAT TTG3 '; L5'TTA GCT ACC CCC AAG TGT3'. PCR amplification system 50 μL, including the 3 'and 5' end of each primer, 12.5 pmol , 10 × PCR buffer 5 μL, 75 mmol MgCl2, 40 nmol dNTPs, 33.3 nKat Taq DNA polymerase and 1 ng DNA template. deionized water make up 50 μL. loop parameters are at 95 ℃ 3 min 20 s, 94 ℃ denaturation 30 s , 55 ℃ annealing 30 s, 72 ℃ extension of 1.5 min, 35 cycles, 72 ℃ for 7 min (PE9600). PCR products with 10 g · L-1 agarose gel electrophoresis 10 min, post-test with a Wizard purification kit ( Promega Corporation USA) was purified. 1.2.3 and sequencing sequencing PCR reaction volume of 20 μL, including primers, 1.3 μL (2.5 mmol · L-1), mixture of 8.0 μL, purified PCR products were 200 ng. cycle parameters for the 96 ℃ denaturation 10 s, 50 ℃ annealing 5 s, 60 ℃ extension 4 min, 25 cycles. sequenced products were precipitated with the ABI PRISM 377 automated sequencer and sequencing gel electrophoresis, obtained samples of mtDNA sequences of each. SeqEdv1.0.3 compared with the standard sequence comparison software and Anderson. 2 Results in the mtDNA HVS Ⅰ 16091 ~ 16418 Sequence comparison between and Anderson were found in 66 mutation (Tab 1). 98 individuals a total of 396 point mutations, an average of 4 points each individual mutation. types of base transition mutation 346, accounting for 87% of mutation; 44 transversion, accounting for 11% of mutation; base into 1%; base deletion 0.5% (Tab 2.) hot spot mutation site in 16183,16189,16223 and 16362, the mutation frequencies were 19%, 21%, 60% and 43% (Tab 3). 16180 ~ 16194 between the 10 different sequence types (Tab 4), of which 62.2% with the standard sequence. 98 cases were found in 62 samples of haplotypes (Tab 1), in accordance with the degree of genetic variation [2] h = (1-Σx2 ) n / (n-1) (x for the haplotype frequency, n is the number of samples) calculated genetic differences Baoan District HVS Ⅰ degree 0.9862, in accordance with the coupling probability [2] p = Σx2 coupling calculation of the probability of Baoan District, HVS Ⅰ was 0.0237.