本文選題：單核苷酸多態(tài)性(SNPs) + PCR-序列特異性引物(PCR-SSP)��； 參考：《中國醫(yī)科大學(xué)》2004年碩士論文

【摘要】： 前言 人類白細(xì)胞抗原(human leukocyte antigen，HLA)編碼基因位于人類第6號(hào)染色體短臂(6p21)上。1958年，由Dausset確立了第一個(gè)人類白細(xì)胞抗原Mac，它標(biāo)志著HLA研究的開始。HLA系統(tǒng)之所以引起法醫(yī)學(xué)的重視在于它的高度多態(tài)性。HLA每個(gè)基因座上均有眾多的復(fù)等位基因，而且共顯性遺傳，各個(gè)基因座的基因隨機(jī)組合，形成HLA血型系統(tǒng)高度的遺傳多態(tài)性。由于HLA多態(tài)性是由單個(gè)堿基差異造成的序列多態(tài)性(即單核苷酸多態(tài)性)，各個(gè)位點(diǎn)堿基組合又是隨機(jī)的，故人群中HLA基因型可達(dá)10~8種之多，這為法醫(yī)學(xué)個(gè)人識(shí)別和親子鑒定提供了理想的遺傳標(biāo)記。所謂單核苷酸多態(tài)性(single nucleotide polymorphisms，SNPs)是指基因組內(nèi)特定核苷酸位置上存在著兩種不同的堿基，其中最少的一種在群體中的頻率不少于1％。而HLA上SNPs則要高的多。據(jù)估測，人類整個(gè)基因組內(nèi)核苷酸的多態(tài)性分布頻率為0.08％-0.2％，而位于HLA-Ⅰ類基因的核苷酸多態(tài)性分布頻率竟高達(dá)8.6％。所以HLA基因單核苷酸多態(tài)性的研究成為目前法醫(yī)學(xué)研究新的熱點(diǎn)。 多個(gè)SNPs位點(diǎn)的同時(shí)檢測，即SNPs位點(diǎn)的復(fù)合擴(kuò)增，是PCR技術(shù)上的一大進(jìn)步，一次復(fù)合擴(kuò)增同時(shí)獲得多個(gè)SNPs位點(diǎn)的信息，提高了個(gè)人識(shí)別能力，對微量檢材鑒定特別有價(jià)值，尤其適用于法醫(yī)學(xué)檢案。IMS-JST011107、IMS-JST011109、IMS-JST011110是位于JSNP數(shù)據(jù)庫內(nèi)的HLA-DRA基因座上的3個(gè)SNPs位點(diǎn)。HLA-DRA基因是HLA-Ⅱ類抗原重鏈基因之一，有3個(gè)等位基因，由4個(gè)外顯子組成。這三個(gè)SNPs位點(diǎn)位于第三和第四外顯子之間。2000年日本學(xué)者利用基因組掃描結(jié)合序列測定的方法，首次獲得了HLA-DRA基因座3個(gè)SNPs位點(diǎn)的等位基因頻率分布數(shù)據(jù)。國內(nèi)尚無有關(guān)這三個(gè)SNPs位點(diǎn)的群體遺傳學(xué)數(shù)據(jù)。本研究利用PCR-SSP技術(shù)復(fù)合擴(kuò)增HLA-DRA基因座的3個(gè)SNPs位點(diǎn)，調(diào)查了它們在中國北方漢族群體中等位基因分布頻率，，為它們在法醫(yī)學(xué)個(gè)人識(shí)別和親子鑒定中的實(shí)際應(yīng)用提供了必需的基礎(chǔ)數(shù)據(jù)，并探討在法醫(yī)學(xué)領(lǐng)域應(yīng)用的價(jià)值。 材料與方法 本研究應(yīng)用PCR一SSP技術(shù)復(fù)合擴(kuò)增HLA一DRA基因座上3個(gè)SNPs位 點(diǎn)IMS一JSIDlllO7、IMS一JS功11109和IMS一JS，ID 11110，以聚丙烯酞胺凝膠電 泳分離擴(kuò)增產(chǎn)物結(jié)合銀染顯帶的方法，對170例無血緣關(guān)系的中國北方漢 族個(gè)體進(jìn)行多態(tài)性分析。摸索IMS一JS功1一107、IMs一Js功一1 209和IMs- Js功11110位點(diǎn)的復(fù)合擴(kuò)增的最適條件，并在親子鑒定案例中應(yīng)用。 結(jié)果 在退火溫度為58‘’C，兩個(gè)PCR復(fù)合擴(kuò)增體系在xMS一JS功2 1 1 07、xMS- JS功川09和IMS一JS功1 1 1 10位點(diǎn)引物濃度比分別為0.4/0.6/1 .0(林mol/ L)和0.8/0.8/0.8(林m0FL)時(shí)，復(fù)合擴(kuò)增產(chǎn)物的電泳譜型清晰，達(dá)到了基 因分型的目的。 IMS一JS功1 1 107位點(diǎn)在170例中國北方漢族個(gè)體共檢出由兩個(gè)等位基 因構(gòu)成的三種基因型:C/T、C/C、T/T.其中T/T為巧例，頻率為0 .0882;C/ T為73例，頻率為0.4249;C/C為82例，頻率為0.4823;等位基因C頻率為 O‘6971，等位基因T頻率為0.3029。IMS一JS功1 1 109位點(diǎn)檢出由兩個(gè)等位 基因構(gòu)成的三種基因型:C/T、C/C、T/T.其中T/T為12例，頻率為0.0706; C/T為84例，頻率為0.4941;C/C為74例，頻率為0.4353;等位基因C頻 率為0.6823，等位基因T頻率為0.3176。IMS一JS功11110位點(diǎn)檢出由兩個(gè) 等位基因的構(gòu)成三種基因型:C/A、A/A、C/C。其中燈A為n例，頻率為 0.0647;C/A為83例，頻率為0.4882。C/C為76例，頻率為0.4470:等位 基因C頻率為0.6912，等位基因A頻率為0.3088(表3)。此外，由于本研 究采用單側(cè)的引物，3個(gè)SNPs位點(diǎn)均未檢測到新的變異。 復(fù)合擴(kuò)增3個(gè)SNPs位點(diǎn)檢測5例親子鑒定案例，有4例雙親均可以提 供給子相應(yīng)的等位基因，1例父親不能提供給子相應(yīng)的等位基因。案例1 在IMS一JS，ID 1 1 107位點(diǎn)父、子、母的基因型分別為C/C、T/T、T/T。父不能提 供給子等位基因T。其中案例1、2的復(fù)合擴(kuò)增圖譜如(圖8)所示，案例1- 5的基因型檢測結(jié)果見(表4)。 討論 本研究成功地摸索了IMS一JS，ID 1 1 107、IMS一JS，ID 1 1 109和IMS一JS功1 1 1 10 三個(gè)位點(diǎn)復(fù)合擴(kuò)增的條件，且效果良好，提高了個(gè)人識(shí)別能力，具有快速、鑒 別率高、節(jié)省檢材等優(yōu)點(diǎn)，可以用于個(gè)人識(shí)別和親子鑒定案例。 HLA一DRA基因座3個(gè)SNPs位點(diǎn)在中國北方漢族群體中的DP值分別 為0.5674、0.5664、0.5577;EPP值分別為0.1666、0.1704、0.1679;PIC值分 別為0.4223、0.4335、0.4269;H值分別為0.4294、0.4941、0.4882。CDP為 0.9165，eEPP為0.4247，累積Pxe為0.8124(表5)。這三個(gè)位點(diǎn)都是具有 高鑒別能力的遺傳標(biāo)記系統(tǒng)，在法醫(yī)學(xué)個(gè)人識(shí)別和親子鑒定中具有很高的 應(yīng)用價(jià)值。 本研究獲得的中國北方漢族群體IMS一JsTDI 1 107、IMS一JS功1 1109和 IMS一JS功11110三個(gè)SNPS位點(diǎn)的基因頻率分布結(jié)果與國外報(bào)道的日本人 群的頻率分布進(jìn)行x，檢驗(yàn)，發(fā)現(xiàn)兩群體之間無顯著性差異(P0.05)(表 6)。這說明了IMS一JS功1 1 107、IMS一JS功11109和IMS一JS功11110位點(diǎn)的等 位基因在中國漢族和日本人群中分布具有同源性，這一現(xiàn)象可以用來解釋 中日兩群體具有共
[Abstract]:Preface
The human leukocyte antigen (human leukocyte antigen, HLA) coding gene is located on the human chromosome sixth on the short arm (6p21).1958, and the first human leukocyte antigen Mac is established by Dausset, which marks the beginning of the HLA study of the.HLA system that causes forensic medicine to focus on its high polymorphism.HLA on each of the loci. Multiple alleles, and co dominant inheritance, and a random combination of genes in each loci, form a genetic polymorphism of the HLA blood type system. Because HLA polymorphism is a sequence polymorphism (single nucleotide polymorphism) caused by a single base difference (i.e., single nucleotide polymorphisms), and the combination of each site base is random, so the HLA genotype can reach 10~8 species in the population. It provides an ideal genetic marker for forensic personal identification and parent-child identification. Single nucleotide polymorphisms (SNPs) refers to the existence of two different bases on the specific nucleotide sites in the genome, the least of which is less than 1%. in the group and higher in SNPs on HLA. It is estimated that the polymorphism of nucleotide polymorphisms in the whole human genome is 0.08%-0.2%, and the frequency of nucleotide polymorphisms in the HLA- I gene is up to 8.6%., so the study of the single nucleotide polymorphism of the HLA gene has become a new hot spot in forensic research.
The simultaneous detection of multiple SNPs loci, that is, multiplex amplification of the SNPs site, is a major advance in PCR technology. A composite amplification is used to obtain information of multiple SNPs sites at the same time. It improves the individual recognition ability and is of particular value for the identification of trace materials, especially for the forensic case.IMS-JST011107, IMS-JST011109, and IMS-JST011110 is located in JSNP. The 3 SNPs loci.HLA-DRA gene in the HLA-DRA loci in the database is one of the HLA- class II antigen heavy chain genes, with 3 alleles and 4 exons. The three SNPs loci are located between the third and the fourth exon of the Japanese Japanese scholars for the first time using the method of genomic scanning and sequencing. The HLA-DRA gene was obtained for the first time. There is no data on the allele frequency distribution of the 3 SNPs loci. There is no population genetic data about these three SNPs loci. This study used PCR-SSP technology to amplify the 3 SNPs loci of the HLA-DRA loci and investigated the frequency of their secondary gene distribution in the Han population in northern China, for their personal identification and parent children in forensic medicine. The practical application of identification provides the necessary basic data and discusses the value of application in forensic medicine.
Materials and methods
In this study, PCR SSP technique was used to amplify 3 SNPs sites on HLA DRA locus.
Point IMS one JSIDlllO7, IMS one JS work 11109 and IMS one JS, ID 11110, polypropylene phthalamine gel gel
170 cases of unrelated northern Han Chinese were analyzed by the method of combining silver staining and banding.
The individuals were analyzed for polymorphism. IMS 1 JS 107 IMs Js Js and IMs-
The optimal conditions for the multiplex amplification of Js power 11110 loci were applied in paternity testing cases.
Result
At the annealing temperature of 58 '' C, the two PCR multiplex amplification system worked at xMS JS 21107, xMS-
JS, Chuan Chuan 09 and IMS one JS work 11110 site primer concentration ratios were 0.4/0.6/1.0 (Lin mol/)
The electrophoretic pattern of the amplified products was clear and reached the base when L and 0.8/0.8/0.8 (Lin m0FL).
For the purpose of classification.
IMS a JS work 11107 loci detected two alleles in 170 northern Han Chinese individuals.
Because of the three genotypes: C/T, C/C, T/T., T/T is a good example, with a frequency of 0.0882; C/
T was 73 cases, frequency was 0.4249, C/C was 82, frequency was 0.4823, allele C frequency was
O '6971, allele T frequency 0.3029.IMS, JS JS 11109 loci detected by two alleles.
There are three genotypes of genes: C/T, C/C, T/T., of which T/T is 12 and the frequency is 0.0706.
C/T was 84 cases, frequency 0.4941, C/C 74 cases, frequency 0.4353, allele C frequency.
The rate of allele T was 0.6823, and the frequency of 0.3176.IMS allele was 11110. The detection of 11110 loci was two.
Alleles consist of three genotypes: C/A, A/A, C/C., where the lamp A is n, and the frequency is
0.0647, C/A for 83 cases, frequency 0.4882.C/C for 76 cases, frequency of 0.4470: alleles.
The frequency of the gene C was 0.6912, and the allele A frequency was 0.3088 (Table 3).
Unilateral primers were used and no new mutations were detected in 3 SNPs loci.
5 cases of paternity testing were detected by multiplex amplification of 3 SNPs loci, and 4 parents were able to mention it.
The corresponding alleles were supplied to the offspring, and 1 fathers could not provide the corresponding alleles to the offspring. Case 1
At IMS JS, ID 11107 loci, the parent genotype is C/C, T/T, T/T. the father can not mention.
Supply sub allele T., in which case 1,2 composite amplification map, as shown in Figure 8, case 1-
The results of 5 genotypes were found (Table 4).
discuss
This study has successfully explored IMS one JS, ID 11107, IMS JS, ID 11109 and IMS JS 11110.
The three loci amplify the conditions, and the effect is good, which improves the ability of personal identification.
It can be used for personal identification and paternity testing cases.
The DP values of 3 SNPs loci of HLA DRA locus in northern Han population of China are respectively
The values are 0.5674,0.5664,0.5577; EPP values are 0.1666,0.1704,0.1679 and PIC respectively.
Don't be 0.4223,0.4335,0.4269; H value is 0.4294,0.4941,0.4882.CDP respectively.
0.9165, eEPP is 0.4247, and cumulative Pxe is 0.8124 (Table 5). These three loci are all
The highly discriminating genetic marker system is very high in forensic personal identification and paternity testing.
Applied value.
In this study, IMS JsTDI 1107, IMS JS and 11109 JS were obtained from the Han population in northern China.
The gene frequency distribution of IMS 11110 JS three SNPS loci results are compared with those reported in Japan.
There was no significant difference between the two groups in the frequency distribution of X (P0.05).
6). This shows that IMS one JS work 11107, IMS one JS work 11109 and IMS one JS 11110 loci.
The distribution of alleles in Chinese Han and Japanese populations is homologous. This phenomenon can be used to explain.
The two groups of China and Japan have the same

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2004
【分類號(hào)】：D919

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