【摘要】：目的:篩選線粒體DNA(mtDNA)編碼區(qū)頻率較高的單核苷酸多態(tài)性(SNP)位點,研究其在中國漢族人群中的多態(tài)性;建立變性高效液相色譜(dHPLC)技術用于研究mtDNA 編碼區(qū)SNP 的方法;利用所篩選的SNP 為線粒體DNA生物芯片的研究提供信息。 方法:針對文獻報道的線粒體DNA 編碼區(qū)多態(tài)性較高的區(qū)域,分成四段,即nt8162～8483(mtco1)、nt13070～13299(mtco2)、nt10287～10679(mtco3)、nt8507～8805(mtco4),分別設計引物,并且優(yōu)化引物擴增條件,使之能同條件擴增;應用直接測序技術研究100 例中國漢族樣本的mtco1、mtco2 多態(tài)性;隨機抽取15 例樣本,同時用測序和dHPLC 技術分別檢測mtco3、mtco4 的多態(tài)性,在此基礎上比較dHPLC 和測序兩種方法結果的一致性,從而建立dHPLC 技術用于研究mtco3、mtco4 多態(tài)性的方法,再用已經建立的dHPLC 方法檢測其余85 例樣本的mtco3、mtco4 多態(tài)性。 結果:在100 例中國漢族人群中,mtco1、mtco2 共檢出21 種變異,24種單倍型,基因多樣性h 值為75.11%,偶合概率P 值為25.64%,其中,mtco1序列有15 例樣本(15%)在COⅡ/tRNALys之間發(fā)生9bp(CCCCCTCTA)缺失,1 例樣本發(fā)生9bp(CCCCCTCTA)插入;mtco3、mtco4 共檢出23 個SNP 位點,23 種單倍型,基因多樣性h 值為84.14%,偶合概率P 值為16.70%。四段序列聯(lián)合起來,共檢出44個變異位點,42種單倍型,基因多樣性h值為94.79%,偶合概率P 值為6.16%。在所檢測到的SNP 位點中,nt10400 及nt8701 的突變頻率最高,均為57%,nt10398 為38%,nt8584 為20%,nt8414 為19%。 結論:只有不斷擴大mtDNA 的檢測范圍才能提高其個體識別能力,滿足法醫(yī)學鑒定的需要;建立的dHPLC 方法可用于快速、準確地檢測mtDNA編碼區(qū)多態(tài)性,而且與直接測序法結果完全一致。
[Abstract]:Objective: to screen the single nucleotide polymorphism (SNP) loci with high frequency of mitochondrial DNA (mtDNA) coding region, and to study its polymorphism in Chinese Han population, and to establish a denaturing high performance liquid chromatography (dHPLC) technique for the study of SNP in mtDNA coding region. The selected SNP was used to provide information for the study of mitochondrial DNA biochip. Methods: the regions with high polymorphism of mitochondrial DNA were divided into four segments: nt8162~8483 (mtco1), nt13070~13299 (mtco2), nt10287~10679 (mtco3), nt8507~8805 (mtco4). The conditions of primer amplification were optimized so that the primers could be amplified under the same conditions. The mtco1,mtco2 polymorphism of 100 Chinese Han nationality samples was studied by direct sequencing technique. Fifteen samples were randomly selected and the polymorphism of mtco3,mtco4 was detected by sequencing and dHPLC respectively. The results of two methods, dHPLC and sequencing, were compared on the basis of which the method of using dHPLC to study mtco3,mtco4 polymorphism was established. The established dHPLC method was used to detect the mtco3,mtco4 polymorphism in the remaining 85 samples. Results: in 100 Chinese Han population, 21 variants and 24 haplotypes were detected by mtco1,mtco2. The gene diversity h value was 75.11, and the probability of coupling was 25.64. In 15 samples (15%) of mtco1 sequence, 9bp (CCCCCTCTA) deletion occurred between CO 鈪，

本文編號：2317323