线粒体DNA编码区单核苷酸多态性研究

【作者】 郝金萍；

【导师】 郭大玮； 刘雅诚；

【作者基本信息】 山西医科大学 ， 法医学， 2005， 硕士

【摘要】 目的:筛选线粒体DNA(mtDNA)编码区频率较高的单核苷酸多态性(SNP)位点,研究其在中国汉族人群中的多态性;建立变性高效液相色谱(dHPLC)技术用于研究mtDNA 编码区SNP 的方法;利用所筛选的SNP 为线粒体DNA生物芯片的研究提供信息。方法:针对文献报道的线粒体DNA 编码区多态性较高的区域,分成四段,即nt8162～8483(mtco1)、nt13070～13299(mtco2)、nt10287～10679(mtco3)、nt8507～8805(mtco4),分别设计引物,并且优化引物扩增条件,使之能同条件扩增;应用直接测序技术研究100 例中国汉族样本的mtco1、mtco2 多态性;随机抽取15 例样本,同时用测序和dHPLC 技术分别检测mtco3、mtco4 的多态性,在此基础上比较dHPLC 和测序两种方法结果的一致性,从而建立dHPLC 技术用于研究mtco3、mtco4 多态性的方法,再用已经建立的dHPLC 方法检测其余85 例样本的mtco3、mtco4 多态性。结果:在100 例中国汉族人群中,mtco1、mtco2 共检出21 种变异,24种单倍型,基因多样性h 值为75.11%,偶合概率P 值为25.64%,其中,mtco1序列有15 例样本(15%)在COⅡ/tRNALys之间发生9bp(CCCCCTCTA)缺失,1 例样本发生9bp(CCCCCTCTA)插入;mtco3、mtco4 共检出23 个SNP 位点,23 种单倍型,基因多样性h 值为84.14%,偶合概率P 值为16.70%。四段序列联合起来,共检出44个变异位点,42种单倍型,基因多样性h值为94.79%,偶合概率P 值为6.16%。在所检测到的SNP 位点中,nt10400 及nt8701 的突变频率最高,均为57%,nt10398 为38%,nt8584 为20%,nt8414 为19%。结论:只有不断扩大mtDNA 的检测范围才能提高其个体识别能力,满足法医学鉴定的需要;建立的dHPLC 方法可用于快速、准确地检测mtDNA编码区多态性,而且与直接测序法结果完全一致。更多还原

【Abstract】 Objective To explore the new mitochondrial DNA(mtDNA) singlenucleotide polymorphisms(SNP) in coding area;Establishing an effectivetechnique of denaturing high-performance liquid chromatography(dHPLC) toscreen mtDNA SNPs in coding area.To provide a basis for biological chip withthe use of SNP loci.Methods According to the reported polymorphic region ofmtDNA coding area,four segments were determined,including nt8162 ～8483(mtco1)、nt13070～13299(mtco2)、nt10287～10679(mtco3)、nt8507～8805(mtco4),whose primers were designed according to the Anderson’ssequence.The amplification system was optimized so that the different segmentscan be amplified under the same condition.Using PCR-sequencing method todetect SNPs of mtco1 and mtco2 segments in 100 Chinese Han populations.Then 15 samples were selected randomly,whose polymorphisms of mtco3 andmtco4 were determined by sequencing and dHPLC method respectively. ThusdHPLC method can be evaluated and established on the basis of 15 samples’sequencing results of mtco3 and mtco4.The established dHPLC method werethen employed to investigate other sample’s polymorphisms of mtco3 andmtco4.Results Among 100 Chinese Han populations,there were 24 mtDNAhaplotypes caused by 21 variations in mtco1 and mtco2 segments,of which thegenetic diversity(h) was found to be 75.11% and the probability of two unrelatedpersons having the same sequence(P) was 25.64%.In mtDNA COⅡ/tRNALysregion of mtco1,1 sample(1%) have 9bp(CCCCCTCTA) insertion and 15samples(15%) have 9bp deletion.In mtco3 and mtco4 segments,23 haplotypeswere identified,which were caused by 23 variations.The h and P value were84.14% and 16.70% respectively.In combination of the four segments,a total of42 haplotypes were identified,which were caused by 44 variations,of whichnt10400 and nt8701 were the most(57%),then the nt10398(38%),nt8584(20%),nt8414(19%).The h and P value of the combined four segments were 94.79% and 6.16% respectively.Conclusions The extended detecting area of mtDNA should be applied to Forensic science to increase the discriminational power.The established dHPLC method was proved to be effective to apply in detecting mutations.And the detected SNP sites have provided a theretical basis for biological chip.更多还原