【摘要】： 目的：線粒體DNA（mitochondrial DNA,mtDNA）因具有拷貝數(shù)多、母系遺傳、進(jìn)化速度快等特點而被應(yīng)用于法醫(yī)學(xué)個人識別和母系親子鑒定中，尤其對于微量、降解、無核生物檢材提供了一種有效的檢測途徑。mtDNA由編碼區(qū)和非編碼區(qū)構(gòu)成。非編碼區(qū)也叫控制區(qū)（control region,CR）或D-loop(displacement loop)區(qū)，包括三個高變區(qū)：高變區(qū)1（hypervariable region,HV1）、高變區(qū)2（hypervariable region ,HV2）和高變區(qū)3（hypervariable region ,HV3）。其序列多態(tài)性區(qū)域主要集中在HV 1和HV 2，對世界上很多人群的調(diào)查結(jié)果顯示mtDNA HV1、HV2區(qū)具有較高的遺傳多態(tài)性，而編碼區(qū)具有高度保守性。以往認(rèn)為在健康人群中此區(qū)域沒有基因突變，但近來國外有報道編碼區(qū)8389-8865nt和Cyb基因在人群中也具有遺傳多態(tài)性。目前國內(nèi)已有對中國人群的此類報道，但尚未見編碼區(qū)人群多態(tài)性的報道，更未見對河北漢族人群mtDNA多態(tài)性的研究。本研究選取了mtDNA HV1、HV2重疊片段和編碼區(qū)8430-8673nt對100－175名河北漢族無關(guān)個體進(jìn)行遺傳多態(tài)性調(diào)查，同時對10個2代已確定父母子血緣關(guān)系的家系進(jìn)行分析。毛干是現(xiàn)場常見的生物檢材，只含有mtDNA，我們對20例毛干進(jìn)行HV3 WP=4 區(qū)（CA）n重復(fù)子長度多態(tài)性分析。通過以上研究，首次獲得河北漢族人群mtDNA的遺傳學(xué)數(shù)據(jù)，為mtDNA數(shù)據(jù)庫的建立及法醫(yī)學(xué)應(yīng)用奠定了基礎(chǔ)。 方法：采用鹽析法提取新鮮血及腐敗血樣DNA，分別對mtDNA HV1、HV2重疊片段進(jìn)行PCR擴增，6%非變性聚丙烯酰胺凝膠垂直電泳進(jìn)行SSCP分型，硝酸銀染色。對毛干用Chelex法提取DNA，PCR特異擴增，8%非變性聚丙烯酰胺凝膠垂直電泳分型，硝酸銀染色。對樣品逐一分型后，觀察基因型分布，計算各基因型頻率，選取SSCP帶型比較接近，目測判型困難的基因型樣本測序。用excell進(jìn)行統(tǒng)計學(xué)處理，計算遺傳變異度和人群偶合概率。將各基因型頻率分布與國內(nèi)外其他人群進(jìn)行比較。測序結(jié)果用NCBI BLAST與Anderson序列比對，各序列之間用DNASTAR軟件比對。 結(jié)果：在159名河北漢族無關(guān)個體中，HV1A共檢出39個基因型，基因頻率在0.006289-0.09433之間，偶合率P為0.0381，遺傳變異度H為0.9680。在104名河北漢族無關(guān)個體中，HV1B共檢出25個基因型，基因頻率在0.009615-0.1442之間，偶合率P為0.0793，遺傳變異度H為0.9296。在100名河北漢族無關(guān)個體中，HV2A共檢出22個基因型，基因頻率在0.01-0.18之間，偶合率P為0.0914，遺傳變異度H為0.9178。在101名河北漢族無關(guān)個體中，，HV2B共檢出19個基因型，基因頻率在0.009901-0.1881之間，偶合率P為0.0873，遺傳變異度H為0.9218。在175名河北漢族無關(guān)個體中，編碼 WP=5 區(qū)8430-8673nt共檢出29個基因型，基因頻率在0.0057-0.1657之間，偶合率P為0.0936，遺傳變異度H為0.9116。在河北漢族人群100名健康無關(guān)個體中線粒體DNA 5個片段共分出91種單倍型。聯(lián)合HV1、HV2和編碼區(qū)8430－8673基因片段遺傳變異度H為0.9977,偶合概率為0.013297。以上5個位點所選基因型樣本經(jīng)測序證實各序列之間均有差異。HV1、HV2區(qū)各序列與Anderson序列比較，共檢測出65個變異位點，44個與MITOMAP收錄的基因突變相同。其中16223t，16362C，73g，263g, 8563g,8584a等與以往報道的中國漢族其他地區(qū)（北京、山西、哈爾濱、廣州、臺灣）的結(jié)果相符。在98、197、204、316、16168、16184、16188、16195、16266等位置發(fā)現(xiàn)的基因突變與MITOMAP所收錄不同。87、253、317、389、411、16013、16061、16370、8410、8459、8604、8650等位置在MITOMAP中未見收錄。應(yīng)用MITOMAP Mitoanalyzer Tool (NIST) 分析這些突變位置，編碼區(qū)除8410c-t、8604t-c為同義突變，其它均為錯義突變。以上5個位點通過10個家系2代人30名個體的分析發(fā)現(xiàn)孩子的基因型與母親基因型相同，而與父親的不同，表明mtDNA是通過母親傳遞給后代，為母系遺傳。在人群及家系調(diào)查中均未發(fā)現(xiàn)異質(zhì)現(xiàn)象。HV3區(qū)（CA）n重復(fù)子調(diào)查結(jié)果：對20名河北漢族無關(guān)個體提供的毛發(fā)進(jìn)行分析，共發(fā)現(xiàn)3個基因型：4、5、6，重復(fù)次數(shù)在4-6次之間，片段大小為88-92bp，基因型頻率分別為0.3、0.25、0.45，遺傳變異度H為0.678。用Fisher WP=6 確切概率法將河北漢族人與其他人群比較，發(fā)現(xiàn)基因型頻率分布與中國漢族及其他種族間存在顯著性差異。 結(jié)論：HV1、HV2區(qū)重疊片段包含mtDNA D-loop50%以上的點突變，且相同的多態(tài)位點在兩個重疊片段中分別位于不同的位置，通過SSCP分析得到兩種不同的帶型，提高了SSCP檢測的靈敏度。HV1、HV2區(qū)及編碼區(qū)8430－8673區(qū)段片段各長250bp左右，適合SSCP方法檢測。盡管有文獻(xiàn)報道PCR-SSCP方法可檢出90%以上的點突變，但由于SSCP方法電泳影響因素很多（如電壓、凝膠濃度、溫度、緩沖液等），與序列測定相比，SSCP方法檢出的基因型類型很少，這說明SSCP會漏檢一些突變，但測序方法費時、費用昂貴且對實驗室條件有一定要求，SSCP技術(shù)操作相對簡單、快速、成本低、容易掌握，適于大樣本篩查，所以PCR-SSCP技術(shù)可在基層法醫(yī)檢案中應(yīng)用，作為一種初步篩選方法，以節(jié)約時間，降低消耗。 本研究所采用的實驗方法對含量約0.5-1ng/μl的DNA、降解DNA和毛干DNA進(jìn)
[Abstract]:Objective: the mitochondrial DNA (mitochondrial DNA (mtDNA)) has been applied to forensic personal identification and maternal parent-child identification because of its multiple copy number, maternal inheritance and rapid evolution, especially for micro, degradation, and non nuclear biomaterials..mtDNA is composed of the coded region and non coding region. The non coding region is a non coding region. Also called the control area (control region, CR) or D-loop (displacement loop) area, including three high variable zones: high variable zone 1 (hypervariable region, HV1), high variable region 2 (hypervariable region, HV2) and high variable region 3. MtDNA HV1, HV2 region has high genetic polymorphism, and the coding region is highly conserved. It was considered that there was no genetic mutation in this area in healthy people, but recently there have been reports of genetic polymorphism of 8389-8865nt and Cyb genes in the coded regions abroad. The mtDNA polymorphism of the Hebei Han population was not studied. The genetic polymorphism of 100-175 unrelated Hebei Han individuals was investigated by mtDNA HV1, HV2 overlapping fragments and coding region 8430-8673nt. The common biological samples at the scene contain only mtDNA. We performed HV3 on 20 cases of hair shaft.
WP=4
CA n repeats polymorphism analysis. Through the above study, the genetic data of mtDNA in Hebei Han population was obtained for the first time, which laid the foundation for the establishment of mtDNA database and the application of forensic medicine.
Methods: fresh blood and DNA were extracted by salting out. MtDNA HV1, HV2 overlapped fragments were amplified by PCR respectively. 6% non denatured polyacrylamide gel electrophoresis was used for SSCP typing and silver nitrate staining. DNA was extracted by Chelex method, PCR specific amplification, 8% non denatured polyacrylamide gel electrophoresis and silver nitrate staining. After the sample was divided, the genotype distribution was observed, the genotype frequency was calculated, and the SSCP band type was selected to be close, and the genotype samples were sequenced. The genetic variation and the population coupling probability were calculated by excell. The genetic frequency distribution was compared with the other population at home and abroad. The sequencing results were NC BI BLAST and Anderson sequence alignment, each sequence was compared with DNASTAR software.
Results: among 159 unrelated individuals of Hebei Han, 39 genotypes were detected in HV1A, the frequency of gene frequency was between 0.006289-0.09433, the coincidence rate P was 0.0381, the genetic variability H was 0.9680. in 104 unrelated individuals of the Han nationality, and 25 genotypes were detected by HV1B. The gene frequency was between 0.009615-0.1442, the coincidence rate P was 0.0793, and the genetic variation H was H Among 100 unrelated individuals of Hebei Han, HV2A detected 22 genotypes with a genetic frequency of 0.01-0.18, the coincidence rate of P was 0.0914, the genetic variability H was 0.9178. in 101 unrelated individuals in the Han nationality, and 19 genotypes were detected by HV2B, the frequency of the gene frequency was 0.009901-0.1881, the coincidence rate P was 0.0873, and the genetic variability H was 0.9218.. In 175 unrelated individuals from Hebei Han, the code is coded.
WP=5
A total of 29 genotypes were detected in area 8430-8673nt, the frequency of gene frequency was between 0.0057-0.1657, the coincidence rate P was 0.0936, and the genetic variability H was 0.9116. in 100 unrelated individuals of the Hebei Han population, the mitochondrial DNA 5 fragments were divided into 91 haplotypes. The genetic variability of the combined HV1, HV2 and coding region 8430-8673 was 0.9977. The genotype samples of the 5 loci above 0.013297. showed that there were.HV1 differences between the sequences, and the sequence of HV2 region was compared with the Anderson sequence, and 65 mutation sites were detected, 44 were the same as those included in MITOMAP. Among them, 16223t, 16362C, 73g, 263G, 8563g, 8584a, etc. The results of Beijing, Shanxi, Harbin, Guangzhou and Taiwan were consistent. The gene mutations found in 981972043161616816184161881619516266 and other locations, such as the different.872533173894111601316061163708410845986048650 included in MITOMAP, were not included in the MITOMAP. MITOMAP Mitoanalyzer Tool (NIST) was used to analyze these The mutation location, the coding region is 8410c-t, 8604t-c is synonymous mutation, and the others are missense mutations. The above 5 loci through the analysis of 10 families and 2 generations of 30 individuals found that the child's genotypes are the same as the mother genotypes, but different from the father, it shows that mtDNA is passed through the mother to the offspring and is a maternal inheritance. In the population and family survey The results of the.HV3 region (CA) n repeats were not found. The hair of 20 independent Hebei Han individuals was analyzed, and 3 genotypes were found: 4,5,6, the number of repetitions was 4-6 times, the size of the fragment was 88-92bp, the genotype frequency was 0.3,0.25,0.45, and the genetic variability H was 0.678. using Fisher.
WP=6
The exact probability method was used to compare the genotype frequencies of the Han people in Hebei Province with those of other ethnic groups.
Conclusion: the overlap fragment of HV1, HV2 region contains point mutations above mtDNA D-loop50%, and the same polymorphic loci are located at different positions in two overlapped segments, and two different bands are obtained by SSCP analysis. The sensitivity.HV1 of SSCP detection is improved, HV2 area and 8430-8673 segment segments of the coding region are long 250bp, suitable for SSCP. Method detection. Although there are reports that PCR-SSCP method can detect more than 90% point mutations, but because of many factors affecting SSCP electrophoresis (such as voltage, gel concentration, temperature, buffer and so on), the SSCP method has few genotypes compared with sequence determination, which indicates that SSCP will leak some mutations, but the sequencing method is time-consuming and expensive and expensive. SSCP technology is relatively simple, fast, low cost, easy to master and suitable for large sample screening. So PCR-SSCP technology can be used in the primary forensic examination case, as a preliminary screening method, to save time and reduce consumption.
The experimental method used in this study is to degrade DNA and DNA into 0.5-1ng/ with DNA content of about L.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2003
【分類號】：D919

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