Y染色体STR和SNP的研究

发布时间：2018-06-28 19:05

本文选题：Y染色体 + STR　；参考：《四川大学》2003年博士论文

【摘要】： 目的为了提高Y染色体遗传标记的个人识别能力和非父排除能力，寻找新的Y-STR和建立新一代遗传标记Y-SNP的检测方法十分必要。本课题旨在阐明8个新的Y-STR的等位基因结构和单倍型频率，为法医学应用提供基础。建立用普通高效液相色谱仪检测单链寡聚核苷酸的方法，为检测单碱基引物延伸反应的产物提供定量分析基础。建立单碱基引物延伸反应，掌握单碱基引物延伸反应的一般规律，为法医学Y-SNP分析提供基础。方法在数据库中寻找到8个新的Y-STR基因座；利用软件重新设计其中的两个基因座，即DYS443和DYS444的引物。应用PCR方法扩增Y-STR，电泳分型；对所有基因座的等位基因进行测序，构建等位基因分型标准物，按国际法医遗传学会(ISFG)原则命名等位基因。在普通的高效液相色谱仪上，，建立检测合成的单链寡聚核苷酸方法。利用耐热DNA聚合酶催化单碱基引物延伸反应，建立2个Y-SNP的单碱基引物延伸反应。结果等位基因测序结果提示DYS443、DYS453、DYS455、DYS456是简单重复序列Y-STR，而DYS444、DYS448、DYS457、DYS458是复杂重复序列Y-STR；在我们研究的群体样本中，DYS443、 DYS444、DYS448、DYS453、DYS455、DYS456、DYS457、DYS458的基因变异度分别为0.7742、0.7671、0.7453、0.3545、0.0549、0.6988、 0.5058、0.8213。单倍型变异度为0.9991，其个人识别能力和非父排除率也为0.9991。在普通的高效液相色谱仪上，应用反相离子对高效液相色谱法成功地分离了4种长度不同的单链寡聚核昔酸(18、19、20、Zlnt) 以及两种序列不同的19nt单链寡聚核昔酸。用己知浓度的单链寡聚核营酸作为标准品，制作了浓度与峰面积的标准曲线。建立了分析2个丫SNP 的单碱基引物延伸反应，掌握了单碱基引物延伸反应的规律。应用建立的HPLC方法检测单碱基引物延伸反应的产物，并且对产物进行了定量。结论DYS443和DYS444的引物经重新设计后，扩增片段长度缩短，有利于准确分型，结果证明引物设计是成功的。分析了这些基因座的等位基因序列，为按照ISFG的推荐原则进行标准化命名提供了依据。单倍型变异度表明这8个丫STR具有较好的个人识别能力和非父排除率，能够较好地提高Y染色体遗传标记的个人识别和非父排除能力。在普通高效液相色谱仪上，利用离子对反相高效液相色谱方法能够分辨长度、序列不同的单链寡聚核昔酸，其分离度与寡聚核昔酸的序列和长度都有关，为检测单碱基引物延伸反应的产物提供了基础。通过建立2个Y-SNP的 SNuPE反应，了解SNuPE反应的一般规律，揭示单碱基引物延伸反应的关键是选择合适的DNA聚合酶及合适的引物和模板的比例。为丫SNP 应用于法医学提供了方法学基础。
[Abstract]:Objective in order to improve the individual recognition ability and non paternal exclusion ability of Y chromosome genetic markers, it is necessary to find new Y-STR and establish a new generation of genetic marker Y-SNP. The purpose of this study is to clarify 8 new Y-STR allelic structures and haplotype frequencies, and to provide a basis for forensic application. The method of detecting single strand oligonucleotides by Chromatograph provides a basis for quantitative analysis of the product of single base primer extension reaction. The general rule of single base primer extension reaction is established, and the general rule of single base primer extension reaction is grasps, which provides the basis for Y-SNP analysis of forensic medicine. Methods 8 NEW Y-STR loci were found in the data base; The two primers, DYS443 and DYS444, were redesigned by software. The PCR method was used to amplify the Y-STR and electrophoresis typing; the alleles of all the loci were sequenced and the allele standard was constructed, and the alleles were named according to the international forensic genetic association (ISFG) principle. On the ordinary high performance liquid chromatograph, it was established. The single strand oligonucleotide method was detected. 2 Y-SNP single base primer extension reactions were established by using the heat resistant DNA polymerase to catalyze the single base primer extension reaction. The results of allele sequencing showed that DYS443, DYS453, DYS455, DYS456 were simple repeat sequences Y-STR, while DYS444, DYS448, DYS457, DYS458 were complex repeat sequences. In the group samples we studied, DYS443,
DYS444, DYS448, DYS453, DYS455, DYS456, DYS457, DYS458
Gene variability was 0.7742,0.7671,0.7453,0.3545,0.0549,0.6988.
0.5058,0.8213. haplotype variability was 0.9991, its personal recognition ability and non parent exclusion rate.
Also for 0.9991., the reversed-phase ion pair high performance liquid chromatography is used on the ordinary high performance liquid chromatograph.
4 different lengths of single chain oligonucleotide (18,19,20, Zlnt) were successfully separated by spectral analysis.
And two kinds of 19nt single chain oligonucleotides with different sequences.
Acid as a standard product, the standard curve of concentration and peak area was made. 2 SNP were analyzed.
The single base primer extension reaction has mastered the rule of single base primer extension reaction.
The HPLC method was used to detect the products of single base primer extension reaction, and the products were quantified.
Conclusion after DYS443 and DYS444 primers were redesigned, the length of the amplified fragments was shortened.
The results showed that primer design was successful. The alleles of these loci were analyzed.
The gene sequence provides a basis for standardized naming according to the recommendation principle of ISFG. Haplotype
The variation degree indicates that these 8 STR have better personal recognition ability and non father exclusion rate.
It can improve the ability of personal identification and non parent elimination of Y chromosome genetic markers.
On the liquid chromatograph, the ion pair RP HPLC method can distinguish length and sequence.
The separation degree of different single chain oligonucleic acid is related to the sequence and length of oligonucleotide.
It provides a basis for detecting the products of single base primer extension reaction. Through the establishment of 2 Y-SNP
SNuPE reaction, understand the general rule of SNuPE reaction, and reveal the single base primer extension reaction.
The key is to select suitable DNA polymerase and suitable primers and template ratio. For ya SNP
It provides a methodological basis for the application of forensic medicine.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2003
【分类号】：D919

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