本文選題：TAP2基因　切入點：單核苷酸多態(tài)性　出處：《中國醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】： 前言 抗原處理相關轉運體(transporter associated with antigen processing,TAP)蛋白是一種分布于內質網(wǎng)膜上的轉運蛋白,功能是將胞漿內內源性抗原的降解產(chǎn)物轉運至內質網(wǎng)腔。TAP基因于1990年分別由Trowsdale J等和Spies T等同時發(fā)現(xiàn)。1991年WHO的HLA命名委員會將這一新發(fā)現(xiàn)的基因統(tǒng)一命名為TAP基因。TAP基因位于6p21.3,MHCⅡ區(qū)的DQB1和DPB1之間,包括TAP1和TAP2兩個基因。TAP2基因長16.9kb,含有12個外顯子和11今內含子。 2008年,NCBI報道在TAP2基因中發(fā)現(xiàn)了167個SNPs位點,其中有19個位于外顯子。目前,國內外對TAP2基因的研究主要涉及的是疾病發(fā)生發(fā)展機制和免疫相關性方面。而有關TAP2基因在中國遼寧漢族人群的分布及其法醫(yī)學應用價值國內外尚無報道。 本研究選擇TAP2基因第12外顯子中的4個SNPs位點,分別為651密碼子[2073(?)GT(Arg)→(?)GT(Cys),rs4148876]、665密碼子[2115(?)CA(Thr)→(?)CA(Ala),rs241447]、687密碼子[2181(?)AG(stop)→(?)AG(Gln),rs241448]和697密碼子[2213GT(?)(Val)→GT(?)(Val),rs241449],調查這4個SNPs位點在遼寧地區(qū)漢族人群的頻率分布和單體型的構成情況,為人類遺傳學、法醫(yī)學和臨床醫(yī)學等學科提供有價值的參考數(shù)據(jù)。 材料與方法 1、226例靜脈抗凝血采自遼寧地區(qū)漢族無血緣關系的健康獻血者。18例已知親緣關系家系的DNA樣品由中國醫(yī)科大學法醫(yī)血清學教研室提供。 2、應用雙向等位基因特異性擴增(Bi-ASA)技術調查遼寧地區(qū)北方漢族人群TAP2基因4個SNPs位點的分布頻率,并應用于親子鑒定案例。 3、直接計數(shù)法計算TAP2基因4個SNPs位點的等位基因頻率和基因型頻率;Hardy-Weinberg平衡檢驗以及連鎖不平衡分析;應用SPSS11.5統(tǒng)計軟件進行x~2檢驗及Fisher確切概率法來比較不同人群間等位基因頻率分布差異是否具有顯著性;計算雜合度(H)、多態(tài)性信息量(PIC)、個人識別率(DP)和非父排除率(EPP)。 結果 1、在遼寧地區(qū)漢族人群中,TAP2基因651位點檢測出C和T兩個等位基因,C等位基因的頻率為0.9071,T等位基因的頻率為0.0929;兩個等位基因構成C/C、C/T和T/T三種基因型,頻率分別為0.8086、0.1770和0.0044。 665位點檢測出A和G兩個等位基因,A等位基因的頻率為0.6593,G等位基因的頻率為0.3407;兩個等位基因構成A/A、A/G和G/G三種基因型,頻率分別為0.4513、0.4159和0.1327。 687位點檢測出T和C兩個等位基因,T等位基因的頻率為0.6593,C等位基因的頻率為0.3407;兩個等位基因構成T/T、T/C和C/C三種基因型,頻率分別為0.4513、0.4159和0.1327。 697位點檢測出G和T兩個等位基因,G等位基因的頻率為0.6593,T等位基因的頻率為0.3407;兩個等位基因構成G/G、G/T和T/T三種基因型,頻率分別為0.4513、0.4159和0.1327。 2、連鎖不平衡分析結果表明,651分別與665、687和697位點處于連鎖平衡狀態(tài);665、687和697這三個位點之間處于連鎖不平衡狀態(tài)。本研究檢出這4個位點構成的4種單體型:C-A-T-G型、C-G-C-T型、T-A-T-G型和T-G-C-T型,頻率分別為0.6018、0.3053、0.0575和0.0354。 3、建立了一種雙SNPs位點復合擴增分型技術,并對665、687和697三個位點中任意兩個位點的組合進行了同步基因分型研究。 4、在18個2代3口家系中,有2個家系的生物學父子關系在665、687和697位點被否定;18個家系在651位點均未否定生物學父子關系。 討論 本研究應用雙向等位基因特異性擴增技術檢測ZAP2基因4個SNPs位點在遼寧地區(qū)漢族人群中的多態(tài)性分布。即在單管中加入兩對引物進行PCR擴增,其中一對為公用引物,用于擴增含有SNP位點的片段;另兩條為等位基因特異性引物,分別與相應的一條公用引物構成上、下游引物進行擴增反應。兩條特異性引物的3′末端對應于SNP的變異點上,由引物的3′端控制引物的延伸反應,根據(jù)擴增片段的數(shù)目和長度確定該SNP位點的基因型。并通過在兩條等位基因特異性引物的5′末端附加一段由10個G或C組成的“GC尾巴”,提高延伸反應的特異性。 在單個SNP位點分型的基礎上,本研究還探討了一種進行兩個SNPs位點復合擴增的方法。即在一個PCR反應體系中,加入兩對等位基因特異性引物和一對公用引物,根據(jù)擴增片段在電泳圖譜中出現(xiàn)的數(shù)目和位置不同對兩個SNPs位點進行同步基因分型。這一方法提高了SNPs分析的檢測效率,為后續(xù)三個或更多SNPs位點的同步檢測奠定了基礎。 本研究將遼寧漢族人群TAP2基因651、665和687位點等位基因分布調查結果與其他人群的研究報道資料進行比較,發(fā)現(xiàn)在651位點,遼寧漢族人群與津巴布韋人群具有顯著性差異(P＜0.05);在665位點,與臺灣漢族、津巴布韋、高加索和荷蘭人群具有顯著性差異;在687位點,與維吾爾族、津巴布韋和西班牙人群具有顯著性差異。有關TAP2基因697位點頻率分布的資料目前尚未見報道,因此不能進行不同人群之間基因分布的比較。 在4個SNPs位點中,651位點的H、PIC、DP和EPP分別為0.1687、0.1450、0.3149和0.0772;665/687/697位點的H、PIC、DP和EPP分別為0.416、0.3484、0.6088和0.1744。在所檢測的18個2代3口家系中,有2個家系在665/687/697位點排除父權;18個案例在651位點均未否定生物學父子關系。 結論 1、本研究所建立的SNPs雙位點復合擴增技術,可同時對兩個SNPs位點進行基因分型,具有簡單、快速、特異性好的特點。 2、TAP2基因651和665/687/697位點在遼寧漢族群體中具有遺傳多態(tài)性,且符合Hardy-Weinberg平衡。651、665、687位點在不同種群間具有遺傳差異。 3、651位點屬于中等鑒別能力的遺傳標記,665/687/697位點屬于較高鑒別能力的遺傳標記,在法醫(yī)學親子鑒定和個人識別中具有實際應用價值。
[Abstract]:Preface
Transporter associated antigen processing (transporter associated with antigen processing, TAP) is a protein located in endoplasmic reticulum transport protein function is a degradation product of transport to the endoplasmic reticulum cavity.TAP gene in the cytoplasm of endogenous antigen respectively in 1990 by Trowsdale J and Spies T also found that.1991 WHO HLA Nomenclature Committee this will be a new gene named as TAP gene.TAP gene at 6p21.3 between MHC II region of DQB1 and DPB1, including TAP1 and TAP2 two gene.TAP2 gene was 16.9kb long with 12 exons and 11 introns present.
In 2008, NCBI reported 167 SNPs loci were found in the TAP2 gene, including 19 in exon. At present, the domestic and foreign research on TAP2 gene is mainly involved in the development and mechanism of immune related diseases occurred. The TAP2 gene in Liaoning Han population Chinese distribution and medical application of the domestic law there is no report.
This study selected TAP2 gene twelfth exon 4 SNPs loci in the 651 codon, respectively [2073 (?) GT (Arg), (?) GT (Cys), rs4148876], [2115 codon 665 (?) CA (Thr), (?) CA (Ala), rs241447], password 687 [2181 (?) AG (stop), (?) AG (Gln), rs241448] and [2213GT codon 697 (?) (Val), GT (?) (Val, rs241449]), the composition of this survey the frequency distribution of 4 SNPs loci in Liaoning Han population and haplotype, for human genetics, which provides valuable reference data for forensic science and clinical medicine.
Materials and methods
1226 cases of venous anticoagulant were collected from.18 blood donors in Liaoning area. The DNA samples of known families were provided by the Department of forensic serology, China Medical University.
2, the bidirectional allele specific amplification (Bi-ASA) technique was applied to investigate the distribution frequency of 4 SNPs loci of TAP2 gene in northern Han population of Liaoning, and applied to paternity testing cases.
3, the allele frequency and genotype frequency direct counting method to calculate the TAP2 gene of 4 SNPs loci; Hardy-Weinberg balance test and linkage disequilibrium analysis; application SPSS11.5 statistical software for x~2 test and Fisher exact probability method to whether differences in allele frequency distribution between groups compared different significantly; calculation of heterozygosity (H), polymorphism information content (PIC), personal identification rate (DP) and the probability of exclusion (EPP).
Result
1, in Liaoning Han population, two alleles of C and T were detected at 651 loci of TAP2 gene. The allele frequency of C was 0.9071, the frequency of T allele was 0.0929, and two alleles constituted three genotypes of C/C, C/T and T/T, and the frequencies were 0.8086,0.1770 and 0.0044. respectively.
665 loci detected two alleles of A and G, the frequency of A allele was 0.6593, the allele frequency of G was 0.3407, two alleles constituted three genotypes of A/A, A/G and G/G, and the frequencies were 0.4513,0.4159 and 0.1327. respectively.
687 loci detected two alleles of T and C, the frequency of T allele was 0.6593, the allele frequency of C was 0.3407, two alleles constituted three genotypes of T/T, T/C and C/C, and the frequencies were 0.4513,0.4159 and 0.1327. respectively.
697 loci detected two alleles of G and T, the frequency of G allele was 0.6593, the allele frequency of T was 0.3407, two alleles constituted three genotypes of G/G, G/T and T/T, and the frequencies were 0.4513,0.4159 and 0.1327. respectively.
2, the linkage disequilibrium analysis showed that 651 and 665687 respectively and 697 loci in linkage equilibrium; between 665687 and 697 of the three loci in linkage disequilibrium. The detection of these 4 loci of 4 haplotypes: C-A-T-G type, C-G-C-T type, T-A-T-G type and T-G-C-T type, frequency respectively. 0.6018,0.3053,0.0575 and 0.0354.
3, a double SNPs loci composite amplification typing technology was established, and the synchronous genotyping of 665687 or 697 three loci with any two loci was studied.
4, in the 18 2 generation and 3 families, 2 families were denied the biological father son relationship at 665687 and 697 sites; 18 families did not deny biological father son relationship at 651 sites.
discuss
The research and application of bidirectional allele specific amplification technology to detect the ZAP2 gene of 4 SNPs loci in Liaoning Han population polymorphism distribution. Adding two pairs of primers in a single tube PCR amplification, one of the common primers containing SNP sites for amplification of fragments; another two for allele specific primers were formed with a corresponding common primers, amplification primers. The 3 'end of the corresponding two specific primers for SNP point mutation, by primer extension reaction control primer 3' end, according to the number and length of the amplified fragment to determine the genotype SNP loci. By adding a section consisting of 10 G or C "GC tail" in the 5 'end of the two allele specific primers, increase specific extension reaction.
Based on single SNP locus typing, this study also discusses one of the two SNPs loci multiplex PCR method. In a PCR reaction system, adding two allele specific primers and a pair of common primers according to the amplified fragment appeared in electrophoresis in the number and position of different of the two SNPs loci were simultaneous genotyping. This method improves the detection efficiency of SNPs analysis, which laid the foundation for the subsequent simultaneous detection of three or more SNPs sites.
The distribution of TAP2 gene in Liaoning Han population and 651665 alleles in 687 loci survey results and other population studies reported data were compared, found in 651 loci in Liaoning Han population and Zimbabwe population have significant difference (P < 0.05); in 665 sites, and the Han nationality in Taiwan, Zimbabwe, the Caucasus and Holland people have significant differences; in 687 sites, and Uygur, Zimbabwe and Spain, the crowd has significant difference. The TAP2 697 gene frequency distribution of the data has not been reported, so the comparison of gene distribution between different groups.
In 4 SNPs loci, 651 loci H, PIC, DP and EPP were 0.1687,0.1450,0.3149 and 0.0772 respectively; 665/687/697 loci H, PIC, DP and EPP were 0.416,0.3484,0.6088 and 0.1744. in the 18 generation 2 3 families detected in families with 2 excluding paternity at 665/687/697 loci; 18 A case in 651 sites allthe18caseswerenotexcludedfromthepaternity.
conclusion
1, the SNPs double site multiplex amplification technology established in this study can genotyping two SNPs loci simultaneously, which has the characteristics of simple, rapid, and good specificity.
2, the 651 and 665/687/697 loci of TAP2 gene have genetic polymorphism in Liaoning Han population, and Hardy-Weinberg balance.651665687 loci have genetic differences among different populations.
The 3651 locus is a genetic marker with moderate discriminating ability. The 665/687/697 locus is a genetic marker with high discriminating ability. It has practical application value in forensic paternity testing and personal identification.

【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R394;D919

【共引文獻】

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