本文選題：X染色體 + 短串聯(lián)重復(fù)序列�。� 參考：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:人類X染色體STRs是指存在于X染色體上特異堿基短串聯(lián)重復(fù)片段。近年來X染色體STRs因其高穩(wěn)定性,高多態(tài)性,獨特的遺傳方式,在解決特殊案件中無法比擬的優(yōu)點而逐漸受到重視,雖然早在1991年就有HPRTB,ARA和DXS981等X-STR基因座的報道,但當(dāng)前已獲得群體遺傳學(xué)數(shù)據(jù)且可用于法醫(yī)遺傳學(xué)分析的X-STR基因座僅有30多個,X染色體STRs群體遺傳學(xué)數(shù)據(jù)嚴(yán)重欠缺,數(shù)量不足,存在人群和種族差異,基因座間連鎖關(guān)系又限制其使用,因此研究尋找更多能為法醫(yī)學(xué)所使用的X-STR基因座,顯得尤為重要。 本課題旨在選取4個未報道群體遺傳特征的X-STR基因座,即GATA151A05/GATA2B05/DXS7129/ HUMUT1595,并構(gòu)建熒光標(biāo)記復(fù)合擴(kuò)增體系,對北方漢族人群進(jìn)行群體遺傳學(xué)調(diào)查,為開發(fā)國產(chǎn)X-STRs試劑盒、DNA數(shù)據(jù)庫建立和群體遺傳學(xué)研究提供基礎(chǔ)資料。 方法: 1基因座的選擇:選擇未報道群體遺傳學(xué)特征且位于X染色體上物理距離較近(10Mb),位于同一連鎖群內(nèi)的4個X-STR基因座。 2復(fù)合分型體系的構(gòu)建:對GATA2B05、HUMUT1595、GATA151A05、DXS7129這4個已選擇的X-STRs進(jìn)行PCR復(fù)合擴(kuò)增。在GATA2B05、HUMUT1595基因座的上游引物5’端標(biāo)記6-FAM熒光,GATA151A05基因座的上游引物5’端標(biāo)記HEX熒光,DXS7129基因座的上游引物5’端標(biāo)記ROX熒光。優(yōu)化各基因座引物濃度比例、Mg2+和Taq酶濃度、退火溫度、循環(huán)次數(shù)等因素。應(yīng)用ABI310及ABI3130型遺傳分析儀對擴(kuò)增產(chǎn)物進(jìn)行檢測,Genemapper 3.2軟件分析結(jié)果并對樣本進(jìn)行檢測分型。各基因座每一種等位基因片段隨機(jī)選擇一個男性樣本進(jìn)行測序,依據(jù)測序結(jié)果并根據(jù)國際法醫(yī)遺傳學(xué)會(international society of forensic genetics, ISFG)推薦的命名原則對各等位基因進(jìn)行命名。 3群體遺傳學(xué)調(diào)查和法醫(yī)學(xué)應(yīng)用:應(yīng)用已復(fù)合的擴(kuò)增體系對北方漢族214例健康無關(guān)個體(男105例,女109例)進(jìn)行群體遺傳學(xué)調(diào)查。結(jié)果用Arlequin3.11軟件進(jìn)行分析,使用http://www.chrx-str.org網(wǎng)站在線計算相關(guān)法醫(yī)遺傳學(xué)參數(shù)。并對本室日常檢案中的10例單親鑒定案件(4例父女單親鑒定案件,6例母子單親鑒定案件)進(jìn)行了調(diào)查,及本法醫(yī)病理教研室尸檢標(biāo)本進(jìn)行了組織同一性檢驗。 結(jié)果: 1熒光復(fù)合分型體系構(gòu)建:成功建立了GATA151A05 /GATA2B05 /DXS7129 / HUMUT1595四個X-STR基因座熒光標(biāo)記復(fù)合擴(kuò)增體系。20μl反應(yīng)體系中含引物GATA151A05、GATA2B05、DXS7129、HUMUT1595分別為6pmol、5pmol、4pmol、5pmol,Taq DNA聚合酶1U,MgCl2濃度1.5mM,采用熱啟動的方式進(jìn)行擴(kuò)增。該體系各基因座間峰型均勻、雜合子擴(kuò)增均衡、無非特異性擴(kuò)增、重復(fù)性好。 2群體遺傳學(xué)調(diào)查結(jié)果:應(yīng)用該體系對北方漢族214例無關(guān)個體進(jìn)行了群體遺傳學(xué)調(diào)查。女性109例樣本中,基因座DXS7129檢出5個等位基因和8種基因型;GATA151A05檢出5個等位基因和9種基因型;HUMUT1595檢出8個等位基因和14種基因型;GATA2B05檢出8個等位基因和18種基因型;男性105例樣本中,基因座DXS7129檢出5個等位基因;GATA151A05檢出6個等位基因;HUMUT1595檢出7個等位基因;GATA2B05檢出8個等位基因。對各基因座進(jìn)行哈溫平衡檢驗,除GATA2B05基因座不符合哈溫平衡外,其余三個基因座均符合哈溫平衡。對符合哈溫平衡的DXS7129,GATA151A05,HUMUT1595基因座進(jìn)行了測序,男女等位基因頻率差異性檢測,基因座間連鎖不平衡分析和遺傳參數(shù)計算。這3個基因座各等位基因在男女之間無差別,合并計算等位基因頻率(0.00309—0.7678),基因座連鎖不平衡分析顯示3個基因座之間相互獨立。3個X-STR基因座DXS7129、HUMUT1595、GATA151A05在北方漢族人群中觀察值雜合度(Ho)分別依次為:0.29358、0.60550、0.54128,多態(tài)信息量(PIC)分別依次為:0.36858、0.62258、0.58478,女性個體排除率(PDfemale)分別依次為:0.593145、0.761857、0.731926,男性個體排除率(PDmale)分別依次為:0.385692、0.602436、0.573134 ,三聯(lián)體非父平均排除概率(MEC Kishida)分別依次為: 0.356211、0.522350、0.487274,二聯(lián)體非父平均排除概率(MEC Desmarais Duo)分別依次為: 0.226958、0.380390、0.346576。 3家系調(diào)查:在對本室日常檢案中的10例單親鑒定案件(4例父女單親鑒定案件,6例母子單親鑒定案件)的檢測中未發(fā)現(xiàn)基因座突變,且這四個X-STR基因座是按照X染色體標(biāo)記物特有的交叉遺傳方式遺傳。 4組織同一性檢測:該體系對同一個體的心臟、肝臟、腎臟、肌肉組織檢測結(jié)果分型一致。 結(jié)論:本研究建立了一組法醫(yī)學(xué)遺傳標(biāo)記GATA151A05 /GATA2B05 /DXS7129 HUMUT1595四色熒光標(biāo)記復(fù)合擴(kuò)增體系,該體系分型準(zhǔn)確、重復(fù)性好。對該體系的群體遺傳學(xué)調(diào)查表明: HUMUT1595具有高度多態(tài)性,GATA151A05具有中度多態(tài)性,可用于法醫(yī)學(xué)親子鑒定和個人識別,充實了尚欠缺的X-STRs群體遺傳學(xué)數(shù)據(jù),可為開發(fā)國產(chǎn)X-STRs試劑盒、建立X-STRs數(shù)據(jù)庫建立提供基礎(chǔ)資料。
[Abstract]:Objective: human X chromosome STRs refers to the specific base short tandem repeats existing on the X chromosome. In recent years, the X chromosome STRs has been gradually valued for its high stability, high polymorphism and unique genetic method, which is unparalleled in solving special cases, although the X-STR loci such as HPRTB, ARA and DXS981 have been reported early in 1991. But there are only more than 30 X-STR loci that have obtained population genetic data and can be used for forensic genetic analysis. The X chromosome STRs population genetic data is deficient, the number is insufficient, the population and race differences exist, and the interloci linkage relationship restricts its use. This study seeks to find more X-STR bases used for forensic medicine. Because of the seat, it is particularly important.
The purpose of this project is to select 4 X-STR loci, that is, GATA151A05/GATA2B05/DXS7129/ HUMUT1595, which is the genetic feature of the unreported population, and to construct a fluorescence labelled composite amplification system to investigate the population genetics of the Han population in the north, and provide the basic information for the development of domestic X-STRs kits, the establishment of DNA database and the study of population genetics.
Method:
1 loci selection: select the unreported population genetic characteristics and the physical distance on the X chromosome is close (10Mb), located in the same linkage group of 4 X-STR loci.
2 the construction of the 2 compound typing system: PCR composite amplification of 4 selected X-STRs for GATA2B05, HUMUT1595, GATA151A05, DXS7129. In GATA2B05, the upstream primers of the HUMUT1595 loci are labeled 6-FAM fluorescence, the upstream primer 5 'of the GATA151A05 loci are labeled HEX fluorescence, and the upstream primers of the DXS7129 genre are labeled with the 5' end of the DXS7129 genre. The ratio of primer concentration, Mg2+ and Taq enzyme concentration, annealing temperature, and cycle times were optimized. ABI310 and ABI3130 genetic analyzer were used to detect the amplification products, Genemapper 3.2 software analysis results and samples were detected. Each allele fragment of each loci was randomly selected for a male sample. Sequenced, the alleles were named according to the sequencing results and based on the naming principles recommended by the international society of forensic genetics (ISFG).
The 3 population genetics survey and forensic application: the population genetics survey of 214 unrelated health individuals (105 men and 109 women) of the Han nationality in the north of the northern Han Dynasty was investigated with a compound amplification system. The results were analyzed by Arlequin3.11 software, and the relevant forensic genetic parameters were calculated online by the http://www.chrx-str.org website. 10 cases of single parent identification (4 cases of paternity single parent identification, 6 parent parent parent identification cases) were investigated, and the autopsy specimens of this forensic pathology teaching and research department were tested for the same identity.
Result:
The construction of 1 fluorescent composite typing system: the GATA151A05 /GATA2B05 /DXS7129 / HUMUT1595 /DXS7129 / HUMUT1595 fluorescent labeling complex amplification system was established, and the primers GATA151A05, GATA2B05, DXS7129, HUMUT1595, respectively, were used as the thermal startup method. The amplify of the loci was homogeneous, heterozygote amplification was balanced, and the amplification was not only non specific amplification, but also good reproducibility.
The results of the 2 population genetics survey: using this system to investigate the population genetics of 214 unrelated individuals in the northern Han population, 5 alleles and 8 genotypes were detected in 109 female samples, 5 alleles and 9 genotypes were detected by GATA151A05, and 8 alleles and 14 genotypes were detected by HUMUT1595; and 8 was detected by GATA2B05. 8 18 alleles and 5 alleles were detected in 105 male samples, 5 alleles were detected by DXS7129, 6 alleles were detected by GATA151A05, 7 alleles were detected by HUMUT1595, and 8 alleles were detected by GATA2B05. The rest of the gene pedestal, except that the GATA2B05 locus was incompatible with the hash balance, had the other three loci. The DXS7129, GATA151A05, HUMUT1595 loci were sequenced, the allele frequency differences of male and female alleles, interloci linkage disequilibrium analysis and genetic parameters calculation. The alleles of the 3 loci were not different between men and women, and the frequencies of alleles (0.00309 - 0.7678) were combined (0.00309 - 0.7678). The linkage disequilibrium analysis showed that the independent.3 X-STR locus DXS7129, HUMUT1595, and GATA151A05 in the northern Han population were respectively 0.29358,0.60550,0.54128, and the polymorphic information (PIC), respectively, was 0.36858,0.62258,0.58478, and the female individual exclusion rate (PDfemale) was respectively dependent on the 3 loci. The following is: 0.593145,0.761857,0.731926, the male individual exclusion rate (PDmale), respectively, is 0.385692,0.602436,0.573134, and the non parent average exclusion probability (MEC Kishida) of the three body (MEC Kishida) is respectively 0.356211,0.522350,0.487274, and the non parent average exclusion probability (MEC Desmarais Duo) of the two body (MEC Desmarais Duo) is respectively 0.226958,0.380390,0.346576..
3 survey: 10 single parent identification cases (4 cases of paternity parental identification, 6 parent parent identification cases) were not detected in the laboratory examination, and the four X-STR loci were inherited according to the characteristic cross inheritance of X chromosome markers.
4 tissue identity test: the system is identical to the heart, liver, kidney and muscle tissue of the same individual.
Conclusion: a group of forensic genetic markers GATA151A05 /GATA2B05 /DXS7129 HUMUT1595 four color fluorescent labeling complex amplification system has been established in this study. The system is accurate and reproducible. The population genetic investigation of this system shows that HUMUT1595 has high polymorphism and GATA151A05 has moderate polymorphism, which can be used in forensic parents. Identification and personal identification have enriched the X-STRs population genetic data which are not yet available. It can provide basic information for developing domestic X-STRs kit and establishing X-STRs database.

【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】：2011
【分類號】：D919

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