【作者】 曾昭书；

【导师】 丁梅；

【作者基本信息】 中国医科大学 ， 法医学， 2005， 博士

【摘要】 目的 自二十世纪60年代以来,法医学界即开始应用微量淋巴细胞毒试验和混合淋巴细胞培养法检测HLA抗原多态性进行法医学亲子鉴定。随着分子生物学技术的迅猛发展和传统抗原分型方法的局限,目前已主要采用基因分型方法分析HLA基因序列多态性来进行法医学亲子鉴定和个人识别,这些方法包括序列特异性引物法、序列特异性寡核苷酸探针法、直接测序分型法以及基因芯片法等,所涉及的基因包括HLA-A、HLA-DRB1以及HLA-DQα等。同传统的血清学、细胞学分型相比,DNA分型准确、迅速、可靠,可避免交叉反应,在法医学鉴定中发挥了重要作用。 HLA-B基因是主要的Ⅰ类基因,序列中SNP位点密集,目前其等位基因数已经达到661种,是多态性最高的基因,在法医学中有较大的应用价值。目前尚缺乏HLA-B特异性SNP位点在中国北方汉族人群中的频率分布资料以及相关法医学应用研究,如果能以特异、灵敏的方法对HLA-B基因序列中的SNP位点进行高通量的分析,获得其群体分布资料,则必将在混合样品检测、非创伤性产前父权鉴定等领域获得重大应用。 因此,本研究的目的是设计出一组HLA-B基因特异的SNP位点探针,应用PCR-SSOP法和PCR-RFLP法对中国北方汉族人群进行频率和家系遗传规律调查,获得相关多态性数据;并将探针应用于原位杂交技术中进行非创伤性产前父权鉴定的尝试,为HLA-B基因多态性的进一步应用奠定基础。 方法 本研究根据已知HLA-B基因序列和各等位基因碱基变异及频率分布的资料,设计2条HLA-B序列特异性DNA探针B009-Z(序列为5’-更多还原

【Abstract】 ObjectiveThe forensic medicine society has used the Complement - dependent Trace Lymphocyte Toxing test and Mixed Lymphocyte Culture ( MLC ) method to detect the Human Leucocyte Antigen (HLA) antigen polymorphism for paternity testing since 1960s. With the rapid advancements of molecular biology and the disadvantages of antigen - typing methods, HLA genotyping nowadays are mainly used instead of antigen - typing. Genotyping methods include sequence - specific primers ( SSP) , sequence - specific oligonucleotide probes ( SSOP) , sequence-based typing (SBT) and microarray, etc. With such methods, genes investigated in forensic field included HLA - A, HLA - DRB1 and HLA -DQA, etc. Compared to the traditional serology typing and cell typing methods, DNA typing methods are exact, fast and trustable, able to avoid cross - reactions, false positives or false negatives. They have played an important role in the real practices.As one of the HLA class I genes, recently HLA - B has been shown to have 661 kinds of sequence alignments along its gene sequence, which are very rich with Single Nucleotide Polymorphism (SNP) mutations. It is thus regarded as the most polymorphic gene among the human genome, and has important application values in forensic medicine field. However, literature retrieval showed that no papers had extended the HLA - B genotyping to forensic medicine field for forensic purpose. So the population data about HLA - B specific SNP mutations among Chinese northern Hans is still in lack, while such a research will be proved very useful in mixed sample detections, non - traumatic prenatal paternity identification and many other forensic applications.So our objectives of this paper were to design a set of HLA - B specific SNP probes to investigate the population data of several selected HLA - B SNP mutations , to find out their heritage characteristics among family lineages, to perform paternity tests with these probes by PCR - SSOP methods, and to explore the possibility of recognizing fetal cells with the probes by In situ Hybridization (ISH) method. Meanwhile, we were also aimed to analyze some HLA SNP frequencies with a PCR — restriction fragment length polymorphism ( PCR — RFLP) method. In general, we expect to establish basises for HLA - B’s forensic applications and for further related researches.MethodsMaking full advantages of the newly published precise sequences of all HLA - B alleles, the clear demonstrations of all the base mutations among HLA - B allele alignments and the allele frequencies about some nationalities and populations, we designed 2 DNA probe sequences, named them as B009 - Z (5’- GG-TATTTCTACACCGCCAT - 3’) and B095 -Z (5’ - ACTTGGCAGACGATG -TATG - 3’) , respectively, all of which can detect several HLA - B gene SNP mutations. By a way of adding betaine to the PCR reaction buffers and following the two - step PCR, hot - start PCR prodecures, we amplified a polymorphic DNA fragment from the HLA - B gene for the SSOP and RFLP analysis. PCR products were dotted onto nylon membranes, blotted with the 2 DNA probes and giving signals. The most optimal SSOP conditions were determined and were further proved by PCR - RFLP, PCR - SSP and DNA sequencing. Samples of 110 unrelated Chinese Northern Hans and 12 families were then detected to find out the distribution frequency and heritage characteristics. Population data was calculated. Also we enriched the peripheral blood mononuclear cells (PBMCs) from the pregnant women s blood, in which the possible fetus’cells - Nucleated Red Blood Cells (NRBCs) were included. PBMC cells were then dispersed onto glass slides with a cell centrifuge. Then we used ISH to detect the slides to recognize which cell was of the fetus origin. At the same time, a SSOP test was performed to both the gradivas and their husbands as controls to ISH results.Suspected fetal cells will be found on the slides when there is no accordance between the ISH signals and dot - blot hybridization signals. Then the possible parentage relation will be inferred.ResultsSuccessful PCR amplifications were achieved with an optimal betaine concentration at 0. 6mol/L, and we got a 943bp DNA fragment from the HLA - B gene sequence, including the most polymorphic region between exon 2 and exon 3. After procedure selections, we determined the most optimal conditions for SSOP as using 4 ui PCR products, hybridization temperature at 46°C and washing temperature at 51°C to both B009 - Z and B095 - Z. PCR - RFLP, PCR -SSP and DNA sequencing methods further proved the hybridization procedures correct, sensitive and specific.Results from SSOP showed that the sequence of Probe B009 - Z had a distribution rate at 0. 545 among Chinese Northern Hans, and B095 - Z at 0.246. In the parentage testing of 12 parentage - certain families with these 2 probes, no clues of parentage exclusions were found. Results from PCR - RFLP with en-donuclease Nlalll showed that a series of HLA - B polymorphic cutting sites exist among non - related Chinese Northern Hans. Totally 6 cutting sites were found, 13 digestion fragments were observed and 20 genotypes were inferred from our 105 samples. Distribution frequencies of the 5 cutting site SNPs were also calculated. We found NRBCs from the gradivas peripheral vein blood by morphological identification under microscope after Wright - Giemsa staining with the slides. While results from ISH with B095 - Z showed that one B095 - Z negative blood sample carried very few B095 - Z positive cells. We thought that the discordance between ISH and SSOP was because the B095 - Z positive cells were of fetus origin, i. e. , the ISH positive SSOP negative cells were from the fetus she carried. In this case no paternity excluded among the gravida, her husband and the fetal cells.更多还原