本文選題：Y染色體 + 短串聯(lián)重復序列��； 參考：《汕頭大學》2009年碩士論文

【摘要】： 背景與目的 人類Y染色體的非重組區(qū)(non-recombination regions of Y chromosome,NRY)為單倍型父系遺傳,這使得Y染色體STR (Y short tandem repeat,Y-STR)在法醫(yī)學個人識別、父權鑒定、男女性混合檢材檢測、無名男尸的身源確定、不同男性個體混合斑或組織混合物的分析,以及群體遷徙、人類進化及父系家族分析中具有獨特的優(yōu)勢,已成為目前法醫(yī)學檢驗的主要遺傳標記。但由于遺傳上的特殊性,與常染色體STR相比,Y-STR單倍型分布具有明顯的群體特異性,且Y-STR的個人識別能力和非父排除率也遠遠低于常染色體STR,要提高Y染色體的鑒別能力,必須盡可能多的聯(lián)合使用Y-STR基因座。目前,國內(nèi)主要依賴進口Y-STR試劑盒分析法醫(yī)物證檢材,其所包含的Y-STR基因座數(shù)量有限,且由于基于國外白種人開發(fā),在一定程度上難以滿足我國法醫(yī)實際工作需要。因此有必要篩選出更多穩(wěn)定、多態(tài)性好、適合我國人群的Y-STR基因座,并構建具有較高鑒定能力的Y-STR復合擴增體系。 本研究旨在篩選更多新的Y-STR基因座,提高Y-STR單倍型的鑒別能力;選取10個新Y-STR基因座,對其中適合MiniSTR引物設計的基因座,進行MiniSTR引物設計;通過對10個Y-STR基因座在潮汕地區(qū)漢族群體中的遺傳多態(tài)性調(diào)查,分析它們的等位基因序列和單倍型,為等位基因命名提供依據(jù),為法醫(yī)學應用提供基礎數(shù)據(jù);根據(jù)群體調(diào)查結果,選出其中符合條件的基因座,應用銀染復合擴增技術,組建Y-STR復合擴增體系,依據(jù)DNA分析技術工作組(the working group on DNA analysis methods,TWGDAM)指南進行法醫(yī)學可行性研究,以建立檢測結果可靠、具有較高個人識別能力的檢測方法。 材料與方法 利用BLAT (BLAST-like alignment tool)軟件對GDB數(shù)據(jù)庫(genome database)中的Y-STR基因座進行種屬特異性分析,依據(jù)具有良好種屬特異性、重復序列為單拷貝、重復單位為四或五核苷酸的原則,篩選出10個新Y-STR基因座DYS522、YS549、DYS556、DYS565、DYS568、DYS570、DYS588、DYS593、DYS594、DYS598;用primer 3.0軟件和BLAT軟件,根據(jù)Y-MiniSTR引物設計原則,對DYS565、DYS568、DYS593、DYS594和DYS598基因座重新進行MiniSTR引物設計,其它基因座引物則采用GDB的引物序列。應用PCR反應,對實驗DNA樣本進行單基因座PCR擴增,擴增產(chǎn)物采用非變性聚丙烯酰胺凝膠連續(xù)緩沖體系垂直電泳分型,銀染法顯色檢測;用女性樣本和常見動物樣本,實驗檢測10個Y-STR基因座的Y染色體特異性和種屬特異性;對潮汕地區(qū)漢族159名無關男性個體樣本進行分型檢測,各基因座的等位基因測序后,按國際法醫(yī)遺傳學會(ISFG)推薦的命名原則命名;采用直接計數(shù)法計算10個Y-STR基因座的等位基因頻率和單倍型頻率;用家系樣本、毛干樣本DNA作為模板,檢測Y-STR基因座的突變率和分析降解DNA的能力。在群體調(diào)查結果基礎上,依據(jù)銀染復合擴增位點選擇的原則,選出符合條件的Y-STR基因座,通過優(yōu)化復合擴增條件,建立復合擴增體系;擴增產(chǎn)物應用非變性聚丙烯酰胺凝膠連續(xù)緩沖體系垂直電泳分型、銀染顯色檢測;用不同DNA含量樣本、不同組織樣本、不同載體上的血痕作模板,對復合擴增體系進行測試。 結果 1 Y-STR特異性分析結果 應用e-PCR對10個基因座進行種屬特異性分析,各基因座的DNA序列只與人類基因組DNA完全匹配,未見其他動物基因組DNA中存在相同序列;用10個Y-STR基因座的引物,擴增女性樣本和常見動物樣本DNA,均未檢測到擴增產(chǎn)物。 2電泳分型 10個Y-STR基因座的擴增產(chǎn)物均只檢測到一條帶,非特異性產(chǎn)物少,分型效果良好。 3等位基因測序結果 10個Y-STR基因座除DYS588和DYS593外,其余均為簡單重復序列Y-STR。它們在潮汕地區(qū)漢族人群中的擴增片段長度在108~372bp之間,其中DYS593擴增片段長度最小,DYS522最大。 4群體遺傳學數(shù)據(jù) 潮汕地區(qū)漢族159名無關男性個體中,DYS522、YS549、DYS556、DYS565、DYS568、DYS570、DYS588、DYS593、DYS594、DYS598基因座分別檢測出7、5、5、4、6、9、8、4、4、4個等位基因,基因變異度在0.1434 (DYS565)~0.7994 (DYS570)之間;10個Y-STR基因座,共觀察到138種不同的單倍型,單倍型變異度為0.9973,標準誤為0.00069。通過30個2代父子家系樣本分析,10個Y-STR基因座單倍型一致,未觀察到突變。 5法醫(yī)學應用結果 10個Y-STR基因座中擴增片段長度小于130bp的DYS565、DYS568、DYS593、DYS598基因座,可對毛干DNA進行檢測分型。 6復合擴增結果 選出了5個Y-STR基因座建立兩組Y-STR復合擴增銀染檢測體系:MultiplexⅠ為DYS549、DYS556和DYS594,MultiplexⅡ為DYS570和DYS593;電泳結果顯示:兩組Y-STR銀染復合擴增體系的各基因座間擴增產(chǎn)物平衡,非變性聚丙烯酰胺凝膠電泳后可對其進行準確分型,且與單基因座擴增結果一致。法醫(yī)學應用研究結果顯示:兩組復合擴增體系具有良好的男性特異性和較高的種屬特異性,最低DNA檢出量達0.5ng;同一個體不同組織器官DNA分型結果一致;對涂在不同載體上的血痕可進行分型,同一樣本分型一致。經(jīng)群體遺傳學數(shù)據(jù)統(tǒng)計,5個基因座在潮汕地區(qū)漢族159名男性樣本中共觀察到97種單倍型,單倍型變異度為0.99,標準誤為0.0013。 結論 1本研究篩選的10個Y-STR基因座都是單拷貝的Y染色體特異性STR基因座,具有很好的種屬特異性和穩(wěn)定的父系遺傳,可用于法醫(yī)學混合斑跡分析和父權關系的鑒定。 2分析了10個Y-STR基因座的等位基因序列,調(diào)查了它們在潮汕地區(qū)漢族人群中的遺傳多態(tài)性,為比較不同群體間的數(shù)據(jù)提供了依據(jù),豐富了我國人類遺傳學數(shù)據(jù)庫。 3 10個Y-STR基因座的單倍型變異度為0.9973,其構成的單倍型具有較高的個人識別率和非父排除率,能夠有效提高Y染色體的鑒別能力。 4證實了小于130bp的小片段MiniSTR可對毛干DNA進行檢測分型,提示對于高度降解的DNA檢材能夠準確分型。 5建立了兩組Y-STR復合擴增銀染檢測體系:MultiplexⅠ和MultiplexⅡ;兩組體系檢測結果可靠,價格低廉;具有很好的男性特異性、種屬特異性、系統(tǒng)重復性,靈敏度高;對附著在各種常見載體上的血痕具有良好的檢測分析能力;個人識別率和非父排除率為0.99,為提高Y染色體的鑒別能力提供了一種經(jīng)濟、快速、高效的檢測方法,可用于法醫(yī)學實際檢案。 6本實驗Y-STR復合擴增體系建立的方法,可作為其他Y-STR基因座銀染和熒光復合擴增的參考依據(jù)。
[Abstract]:Background and purpose
The non recombinant region of the human Y chromosome (non-recombination regions of Y chromosome, NRY) is a haplotype paternal inheritance. This makes Y chromosome STR (Y short tandem) in forensic personal identification, paternity testing, male and female mixed detection, the body source of unnamed male corpse, mixed spots or tissue mixtures of different male individuals. Analysis, population migration, human evolution and paternal family analysis have unique advantages, and have become the main genetic markers of forensic examination. But because of the genetic particularity, the distribution of Y-STR haplotypes has distinct population specificity compared with the autosomal STR, and the individual recognition and non parent exclusion of Y-STR are also far away. Below the autosomal STR, in order to improve the identification ability of the Y chromosome, it is necessary to use as many Y-STR loci as possible. At present, the domestic mainly rely on the import Y-STR kit to analyze forensic material, which contains a limited number of Y-STR loci, and it is difficult to meet the legal practice of our country to a certain extent because of the development of the foreign white people. It is necessary to work. Therefore, it is necessary to screen more stable, polymorphic, suitable Y-STR loci in the population of our country, and to construct a complex Y-STR amplification system with high identification ability.
The purpose of this study is to screen more new Y-STR loci and improve the identification ability of Y-STR haplotype. 10 new Y-STR loci are selected to design the MiniSTR primer which is suitable for MiniSTR primers, and the genetic polymorphism of 10 Y-STR loci in the Han population in Chaoshan region is analyzed, and their allele sequence is analyzed. Column and haplotype, provide basis for allele naming, provide basic data for forensic application, select the suitable loci according to the results of group survey, use silver dye compound amplification technology to form Y-STR compound amplification system, according to the guide of DNA analysis technology working group (the working group on DNA analysis methods, TWGDAM) guide The feasibility study of forensic medicine is to establish a reliable detection method with high personal identification ability.
Materials and methods
The specific analysis of Y-STR loci in GDB database (Genome Database) was carried out by BLAT (BLAST-like alignment tool) software. According to the principle of good species specificity, repeat sequence as single copy and repeat unit as four or five nucleotides, 10 new Y-STR loci were screened for DYS522, YS549, DYS556. YS588, DYS593, DYS594, DYS598; using primer 3 software and BLAT software to design primers for DYS565, DYS568, DYS593, DYS594 and DYS598 genes, and the primers of other loci primers. A non denatured polyacrylamide gel continuous buffer system was used for vertical electrophoresis, silver staining and color detection. The Y chromosome specificity and species specificity of 10 Y-STR loci were tested with female samples and common animal samples, and 159 unrelated male body samples in Chaoshan region were detected by typing, and the alleles of each loci were found. After sequencing, it was named according to the naming principle recommended by the international forensic genetic association (ISFG); the allele frequency and haplotype frequency of 10 Y-STR loci were calculated by direct counting. The mutation rate of Y-STR loci and the ability to analyze the degradation of DNA were detected by family samples and DNA as a template. Based on the results of group investigation, the results were based on the results of group investigation. According to the principle of composite amplification site selection of silver staining, the Y-STR gene pedestal was selected, and the composite amplification system was established by optimizing the complex amplification conditions. The amplified products used the non denaturing polyacrylamide gel continuous buffer system for vertical electrophoresis and silver staining detection, with different DNA content samples, different tissue samples and different carriers. Blood samples were used as templates to test the multiplex amplification system.
Result
1 Y-STR specificity analysis results
The specific analysis of 10 loci was carried out by e-PCR. The DNA sequence of each loci was only completely matched with the human genome DNA, and the same sequence was not found in the other animal genome DNA, and the primers of 10 Y-STR loci were used to amplify the female samples and common animal samples DNA. All the amplified products were not detected.
2 electrophoretic typing
Only one band was detected in the amplified products of the 10 Y-STR loci. The nonspecific products were few and the typing results were good.
3 allele sequencing results
The 10 Y-STR loci, except for DYS588 and DYS593, were all simple repeat sequence Y-STR.. The length of the amplified fragment in the Han population in Chaoshan region was between 108~372bp, of which the length of DYS593 amplification fragment was the smallest and the DYS522 was the largest.
4 population genetic data
Among 159 unrelated male individuals in the Chaoshan region, DYS522, YS549, DYS556, DYS565, DYS568, DYS570, DYS588, DYS593, DYS594, DYS598 loci respectively detected the 7,5,5,4,6,9,8,4,4,4 allele, and the gene variation was 0.1434 (DYS565), and 138 different haplotypes, haplotypes, were observed. The dissimilarity is 0.9973, the standard error is 0.00069.. Through the analysis of 30 2 generation father and son family samples, 10 Y-STR loci haplotypes are identical, no mutation is observed.
5 application results of Forensic Medicine
In the 10 Y-STR loci, DYS565, DYS568, DYS593 and DYS598 loci with a fragment length less than 130bp can be used for detection and typing of hairy stem DNA.
6 compound amplification result
5 Y-STR loci were selected to establish two groups of Y-STR composite amplification silver staining detection system: Multiplex I was DYS549, DYS556 and DYS594, Multiplex II was DYS570 and DYS593. The electrophoresis results showed that the amplification products of the two groups of Y-STR silver staining complex amplification system were balanced, and the non variable polyacrylamide gel electrophoresis could be used to accurately distinguish them. The results of the forensic application study showed that the two groups of composite amplification systems had good male specificity and higher species specificity, and the lowest DNA detection amount was 0.5ng; the DNA typing of different tissues and organs of the same individual was consistent; the blood stains on different carriers could be typed and the same sample was found. According to the data of population genetics, 97 haplotypes were observed by 5 loci in 159 male samples of Han nationality in Chaoshan region. The haplotype variation was 0.99, and the standard was 0.0013.
conclusion
1 the 10 Y-STR loci screened in this study are single copies of the Y chromosome specific STR loci, which have good genera specificity and stable paternal inheritance, which can be used for forensic mixed spot trace analysis and paternity identification.
2 the allelic sequence of 10 Y-STR loci was analyzed and their genetic polymorphism in the Han population in Chaozhou and Shantou area was investigated, which provided a basis for comparing the data between different populations, and enriched the database of human genetics in China.
The haplotype variation of the 310 Y-STR loci was 0.9973. The haplotypes made up of the haplotypes had a high individual recognition rate and a non parent exclusion rate, which could effectively improve the identification ability of the Y chromosome.
4 it was confirmed that the small fragment MiniSTR smaller than 130bp could be used for typing and typing of hairy stem DNA, suggesting that the highly degraded DNA can be accurately typed.
5 set up two groups of Y-STR composite amplification silver staining detection system: Multiplex I and Multiplex II; two groups of system detection results are reliable, low price, and have good male specificity, species specificity, system repeatability, high sensitivity; the blood marks attached to the various common carriers have good detection and analysis ability; individual recognition rate and non The parent exclusion rate is 0.99, which provides an economical, fast and efficient detection method to improve the identification ability of Y chromosome, and can be applied to forensic practice.
6 the method of establishing Y-STR multiplex amplification system can be used as reference basis for silver staining and fluorescence multiplex amplification of other Y-STR loci.
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：D919

【引證文獻】

相關碩士學位論文 前1條

1 李松花;延邊地區(qū)朝鮮族和漢族男性人群六個Y染色體STR基因座的遺傳多態(tài)性[D];延邊大學;2011年

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本文編號：1986075