發(fā)布時(shí)間：2018-02-05 05:53

  本文關(guān)鍵詞： 二核苷酸簡單串聯(lián)重復(fù)順序 D17S518 PCR-STR 毛發(fā)檢材 遼寧漢族　出處：《中國醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 前言 短串聯(lián)重復(fù)序列(short tandem repeats,STR)又稱微衛(wèi)星DNA(mierosatellite),是指重復(fù)單位只有1～6bp、重復(fù)次數(shù)一般在數(shù)次至幾十次之間的串聯(lián)重復(fù)DNA序列。STR在人類基因組中分布廣泛、數(shù)量較多、等位基因片段長度較短、擴(kuò)增成功率高,是目前法醫(yī)物證鑒定中應(yīng)用最廣泛的DNA長度多態(tài)性遺傳標(biāo)記。 然而,對于毛發(fā)等富含角化細(xì)胞的特殊法醫(yī)物證檢材,由于其細(xì)胞核發(fā)生明顯的轉(zhuǎn)移,核基因組DNA含量少且分解嚴(yán)重,常規(guī)方法檢測法醫(yī)物證鑒定中最常用的四核苷酸STR基因座時(shí),常難以獲得滿意的結(jié)果。線粒體DNA(mitochondrialDNA,mtDNA)檢測作為法醫(yī)學(xué)應(yīng)用序列多態(tài)性分析進(jìn)行個(gè)人識別的典型實(shí)例,在毛發(fā)等非常規(guī)檢材的鑒定中具有重要的實(shí)用價(jià)值。但mtDNA特殊的遺傳特征、突變率較高、信息量較低、檢測技術(shù)復(fù)雜等因素的影響,在一定程度上限制了mtDNA的法醫(yī)學(xué)應(yīng)用。因此,選擇多態(tài)性程度較高且等位基因片段更短,適用于毛發(fā)等檢材DNA多態(tài)性分析的遺傳標(biāo)記,是法醫(yī)物證工作者共同關(guān)心的課題。 D17S518基因座位于人類第17號染色體上,是由(CA)n核心序列構(gòu)成的二核苷酸簡單串聯(lián)重復(fù)序列。本研究首先應(yīng)用PCR擴(kuò)增結(jié)合聚丙烯酰胺凝膠電泳分離的方法,調(diào)查D17S518基因座在遼寧漢族人群中的頻率分布,以評估該遺傳標(biāo)記的法醫(yī)學(xué)應(yīng)用價(jià)值,并對其應(yīng)用于毛發(fā)檢材的可行性及影響因素進(jìn)行了初步研究,為該基因座在更廣泛領(lǐng)域的應(yīng)用奠定了基礎(chǔ)。 材料與方法 根據(jù)文獻(xiàn)合成特異性擴(kuò)增D17S518基因座的引物,應(yīng)用PCR技術(shù)和聚丙烯酰胺凝膠電泳分離結(jié)合銀染顯譜的方法,對120例中國遼寧地區(qū)無血緣關(guān)系的漢族個(gè)體血液樣本進(jìn)行D17S518基因座分型,并通過DNA序列分析確定其等位基因;對采用不同方法(酚/氯仿法和Chelex—100法)提取DNA,不同數(shù)量(1根、2根和5根)和不同區(qū)段(遠(yuǎn)段、中段和近段)毛發(fā)檢材的D17S518基因座擴(kuò)增結(jié)果進(jìn)行比較。 結(jié)果 在120例中國遼寧地區(qū)無血緣關(guān)系漢族個(gè)體中,檢出D17S518基因座的5種等位基因及12個(gè)基因型,根據(jù)DNA序列分析結(jié)果將擴(kuò)增片段長度為88bp、90bp、94 bp、98 bp、100 bp的5種等位基因分別命名為D17S518*15、D17S518*16、D17S518*18、D17S518*20和D17S518*21。12種基因型及頻率分別為:15/15為12例(10%),16/16為13例(10.7%),18/1 8為3例(2.5%),15/16為21例(17.5%),15/18為23例(19.2%),15/20為6例(5%),15/21為2例(1.7%),16/18為20例(16.7%),16/20為8例(6.7%),16/21為5例(4.2%),18/20為4例(3.3%),18/21為3例(2.5%);5種等位基因頻率分別為:D17S518*15=0.317、D17S518*16=0.333、D17S518*18=0.233、D17S518*20=0.075、D17S518*21=0.042。應(yīng)用Powerstatsvl2軟件對D17S518基因座群體調(diào)查結(jié)果進(jìn)行數(shù)據(jù)分析,獲得如下法醫(yī)學(xué)應(yīng)用參數(shù):雜合度(H)為0.767,個(gè)人識別幾率(DP)為0.872,偶合幾率(Pm)為0.128,非父排除率(PE)為0.539,多態(tài)信息含量(PIC)為0.68。 6個(gè)個(gè)體毛發(fā)檢材的D17S518基因座擴(kuò)增產(chǎn)物檢測結(jié)果:①采用酚/氯仿方法提取模板DNA,其擴(kuò)增產(chǎn)物檢出率隨毛發(fā)數(shù)量的增加而提高,即1根為2/6、2根為4/6、5根為5/6;而采用Chelex-100法提取模板DNA的毛發(fā)檢材均未檢出擴(kuò)增產(chǎn)物。②同一毛發(fā)不同區(qū)段的擴(kuò)增產(chǎn)物檢出結(jié)果不同;不同毛發(fā)中、遠(yuǎn)段部分?jǐn)U增產(chǎn)物檢出規(guī)律不同,但近段毛發(fā)均可檢出擴(kuò)增產(chǎn)物。 結(jié)論 1、D17S518基因座在中國遼寧地區(qū)漢族群體中具有良好的多態(tài)性分布,在法醫(yī)學(xué)上具有較高的應(yīng)用價(jià)值。 2、二核苷酸簡單串聯(lián)重復(fù)序列的STR基因座適用于毛發(fā)等非常規(guī)法醫(yī)物證檢材的DNA多態(tài)性分析。 3、酚/氯仿法提取毛干DNA的擴(kuò)增效果明顯優(yōu)于Chelex-100法。
[Abstract]:Preface
Short tandem repeat (short tandem, repeats, STR) and microsatellite DNA (mierosatellite), refers to the repeat unit is only 1 ~ 6BP repeat number in general repeat DNA.STR is widely distributed in the human genome, several to dozens of times between the larger number of allele fragment length is short, amplification was successful. High rate, is currently the most widely used in forensic evidence identification DNA length polymorphism genetic markers.
However, for the special forensic samples such as hair in keratinocytes, because of its obvious nuclear transfer, nuclear DNA content and less serious decomposition, conventional methods of forensic identification of tetranucleotide STR loci are most commonly used, often is difficult to obtain satisfactory results. The mitochondrial DNA (mitochondrialDNA, mtDNA) detection as a forensic application of sequence polymorphism analysis of typical examples of personal identification, it has important practical value in the identification of hair and other non conventional materials. But the genetic characteristics of special mtDNA, the mutation rate is higher, the amount of information is low, influence of complex detection technology and other factors, to some extent limit the forensic mtDNA application. Therefore, choose a higher degree of polymorphism and allele for genetic markers in shorter hair samples DNA polymorphism analysis, forensic evidence is the common concern Subject.
D17S518 gene is located on human chromosome seventeenth is composed of (CA) n core sequence consisting of the dinucleotide. In this study we used PCR amplification combined with separation method of polyacrylamide gel electrophoresis, the investigation of D17S518 loci in Liaoning Han population in the frequency distribution, used to assess the genetic markers in forensic. And the feasibility and impact factors of its application in hair samples was studied, which laid the foundation for the application of this locus in a wide range of areas.
Materials and methods
The synthesis of specific primers according to the amplification of the D17S518 locus, separation and silver staining significantly spectrum using PCR and polyacrylamide gel electrophoresis in 120 cases of China unrelated Liaoning Han individual blood samples were D17S518 genotype, and determine its allele by DNA sequence analysis of different methods; (phenol / chloroform method and Chelex 100 method) to extract DNA, different number (1, 2 and 5) and different sections (distal, middle and proximal) D17S518 loci in hair samples were compared.
Result
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本文編號：1492314