应用PCR-RFLP调查武汉汉族人群PGM1基因型

目的PCR RFLP技术调查武汉地区汉族人群PGM 1基因型。方法应用PCR RFLP技术检测PGM 1基因型 ,调查 3 0 0例汉族无关个体。扩增PGM 1基因外显子 4和 8中的靶片段 ,并分别经过Bg1Ⅱ和NlaⅢ限制酶消化。酶切片段经聚丙烯酰胺凝胶电泳分型。结果PGM 1 RFLP技术可分出 9种基因型 ,在汉族人群 ,PGM 1 RFLP系统的个体识别能力为 0 745 0。与传统的PAGE酶型检测比较 ,本法不能区分 1+ 2 -和 1-2 +型 ,不能检测出PGM 1稀有基因 ,但克服了IEF无法分析微量、陈旧材料的缺点 ,对保存 2 5年陈旧血痕及 0 1ng模板DNA均能成功分型。 结论PGM 1 RFLP技术在法医个体识别中有实用价值     

中国4个群体VDR基因2号外显子的SNP基因座遗传多态性

目的 调查中国北方汉族、维吾尔族、藏族和哈萨克族群体维生素D受体基因2号外显子SNP基因座的遗传多态性和群体差别。方法 应用PCR-RFLPs和DNA序列分析技术对271例个体的DNA样品进行分型。结果等位基因ACG的频率最高,分布在0.57～0.72之间。藏族与维吾尔族基因型ACG/ATG的频率超过0.5,汉族基因型ACG/ATG与ACG/ACG的频率均为0.3976;哈萨克族基因型ACG/ACG的频率达到0.5769。DP值与EPP值在4个群体中均超过0.56和0.16。基因型分布符合Hardy-Weinberg平衡,并在4个群体间有一定程度差异。结论 维生素D受体基因2号外显子SNP基因座具有较高的多态性,并具有一定的群体差别。     

D20S161和D8S384两个基因座在法医学中的应用

评估D2 0S16 1和D8S384两个基因座在法医学中的应用价值。用自制的D2 0S16 1和D8S384两个DNA分型试剂盒 ,对人血、人精液、人唾液、动物血、人血与动物血的混合检材和人血痕、人精液斑、人唾液斑、动物血痕、人血与动物血的混合斑痕检材 ,以及陈旧血痕检材进行检测分型 ,并用这两个基因座PCR引物序列与DNA数据库进行联网对比分析。自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血、人精液、人唾液、人血与动物血的混合检材分型 ,而动物血没有PCR产物 ;自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血痕、人精斑、人唾液斑和人血与动物血的混合斑痕检材正确分型 ,而动物血痕没有PCR产物 ;斑痕检材分型结果与对应体液检材分型结果无差异 ;5 0份陈旧血痕检材全部获得阳性分型结果。DNA数据库联网比较提示 ,D2 0S16 1和D8S384基因座引物除了能与各自的模板序列发生特异性扩增外 ,理论上不能与DNA数据库中 6 0 6 36 4种已知序列产生PCR产物。D2 0S16 1和D8S384两个基因座具有高度的种属特异性 ,抗污染能力强 ,不易受降解的影响 ,是解决法医现场生物检材个人识别和亲子鉴定的理想手段     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增（MDA）技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增（WGA）．扩增产物用实时荧光定量PCR技术定量、用Prrfiler Plus^TM试剂盒检测基因型。结果该方法可对模板DNA增加10^4～10^6倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果；低于0．1ng的样品DNA经MDA扩增后，基因座检出数增加。但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0．1ng时，MDA产物的STR分型结果判读须慎重。     

运用二重PCR和DNA芯片技术检测ABO基因型

目的以玻片为载体,用寡核苷酸探针杂交技术检测ABO基因型。方法根据ABO基因座外显子6和外显子7的3个SNP点的序列分布特征设计4条寡核苷酸探针,制成分型芯片。将待测样品DNA用末端标记了Cy5的引物进行二重PCR扩增,产物与芯片上的探针进行杂交,根据杂交产生的荧光信号确定样品的ABO基因型。结果利用ABO芯片,可对血斑、毛发等微量检材进行ABO基因型检测。对115名汉族无关个体的调查表明,ABO基因型的分布符合Hardy-Weinberg平衡,等位基因杂合度观察值和期望值分别为0.591、0.616,多态信息含量为0.544,二联体和三联体非父排除率分别为0.188、0.334,个体识别能力为0.777。结论通过DNA芯片检测ABO基因型的技术适用于法医学样本,可满足高通量的检测需求。     

HLA-DRB1基因分型芯片的法医物证学应用价值研究

目的对HLA-DRB1基因分型芯片在个体识别中的应用价值进行研究。方法根据HLA-DRB1基因座不同等位基因的独特序列设计探针,制成分型芯片。将待测样品DNA用末端标记了CY5的引物进行PCR扩增,产物与芯片进行杂交,根据杂交产生的荧光信号值确定样品在HLA-DRB1位点的基因型。将这一方法应用于561份样本的HLA-DRB1基因分型,根据基因型分布统计分析其法医学应用价值。同时,进行了家系调查和方法灵敏度分析,并应用于部分案例。结果利用微量检材,HLA-DRB1基因芯片可检测DRB1位点等位基因26个,基因型的分布符合Hardy-Weinberg平衡定律,该位点的观察杂合度(Ho)为0.888,期望杂合度(He)为0.902,多态信息含量(PIC)为0.893,平均非父排除率(PE)为0.801。家系调查和案例运用的结果表明,HLA-DRB1位点等位基因由亲代向子代的传递符合孟德尔遗传定律。结论HLA-DRB1为高度多态位点,其基因分型芯片可在亲子鉴定和个体识别中发挥重要作用。     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增(MDA)技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增(WGA),扩增产物用实时荧光定量PCR技术定量、用Profiler PlusTM试剂盒检测基因型。结果该方法可对模板DNA增加104～106倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果;低于0.1ng的样品DNA经MDA扩增后,基因座检出数增加,但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0.1ng时,MDA产物的STR分型结果判读须慎重。     

中国北方汉族群体DRD4基因3个位点遗传多态性

目的调查DRD4基因启动子区-1240L/S、-521C/T和第三外显子48bp VNTR 3个位点在中国北方汉族群体的遗传多态性分布,评价其法医学应用价值。方法收集中国北方汉族207例个体血液样本,提取模板DNA,采用聚合酶链反应和等位基因特异性扩增技术,对3个位点进行分型检测,应用Arlequin 3.5软件对分型数据进行统计分析。结果 3个位点分别检出2个(-1240L/S、-521C/T)和6个(48bp VNTR)等位基因,3种和9种基因型,经χ2检验,各位点基因频率分布均符合Hardy-Weinberg平衡(P0.05);DP值均超过0.5;3个位点共检出14种单倍型,其中8种为主要单倍型,DP值为0.940,PE值为0.804;-1240L/S位点与非洲及高加索人群间的差异具有统计学意义(P0.05),而与日本人群以及-521C/T位点与3种群体之间均无显著性差异(P0.05)。结论中国北方汉族人群DRD4基因3个位点多态性分布均较好,在法医学个体识别与亲子鉴定中具有一定的应用价值。     

两种DNA提取法对不同色泽肋软骨STR分型成功率的比较

目的比较两种DNA提取法对不同色泽肋软骨的DNASTR分型结果的影响。方法利用Chelex-100法和酚-氯仿法,分别对30例不同色泽的腐败尸体肋软骨进行DNA提取,STR复合扩增,ABI3100型基因分析仪对扩增产物进行检测。结果用酚-氯仿法提取的30例腐败尸体肋软骨,均检测到全部STR基因座的等位基因型。用Chelex-100法提取的肋软骨中,22例(11例白色、8例淡黄色、3例黄色)检测出全部STR基因座的等位基因型;7例(3例黄色、4例黄褐色)检测出部分STR基因座的等位基因型;1例黑灰色的腐败尸体肋软骨,未检测出STR基因座的等位基因型。结论根据肋软骨的色泽,选择适宜的DNA提取方法。对于颜色较深的肋软骨,用酚-氯仿法进行DNA提取有助于提高其STR基因座的检出率。     

D8S384基因座的遗传多态性及其在法医物证检验中的应用

揭示人类自然群体中D8S384基因座的基因型频率,评估D8S384基因座在法医物证中的应用价值,以及建立D8S384基因座的分型方法。用不同基因型PCR产物混合的方法,制备了D8S384等位基因分型标准物,并按照国际法医血液遗传学会DNA委员会推荐的原则命名了等位基因。采用PCR扩增、电泳分析、银染显色的方法,调查了世界3大人种11个群体1103名个体的D8S384基因型。D8S384基因座共有8个等位基因,群体内基因型分布符合Hardy－Weinberg平衡,群体间基因型构成有显著性差异。利用群体数据估计了D8S384基因座的法医学理论应用价值,计算得出D8S384基因座的期望杂合度为0．704±0．014,个人识别机率为0．864。D8S384基因座是一个较好的法医学STR遗传标记。     

Analysis of 124 SNP loci included in HID Ampliseq identity panel in a small population of Rio de Janeiro,Brazil

Implementation of massively parallel sequencing platforms can bring a great contribution to Forensic Genetics field, with a great saving of time and costs, as well as allowing reliable results to be obtained from small or extremely degraded samples. The aim of this work was to analyze 124 SNP loci (90 autosomal and 34 Y-SNP) included in HID-Ion Ampliseq Identity Panel in a small sample from Rio de Janeiro state, Brazil. Samples from 12 non-related individuals were amplified with HID-Ion Ampliseq Identity Panel and sequenced on the Ion Torrent PGM platform (Thermo Fisher Scientific); genotypes were generated with HID SNP Genotyper plugin and forensic parameters were calculated with PowerStats v.12. All samples were successfully genotyped and were used to calculate allele frequencies, homozygosity, heterozygosity, random match probability (RMP) and exclusion power for all 90 autosomal SNP loci. Using the formula proposed by Budowle et al. (1996), only 4 of the 90 loci genotyped (4,4%) showed allele frequencies below the minimum required. It means that although a small set of individuals was used on this study, it may have shown a good perspective of Rio de Janeiro state allele frequencies. Among the 11 male samples analyzed, a prevalence of the haplogroup R1b of Y chromosome was observed, followed by the haplogroups E, Q and J. Such distribution reflects the results demonstrated in other studies for the population of Rio de Janeiro. All results together demonstrate the usefulness and applicability of SNP analysis on Ion Torrent PGM.     

红细胞EsD和PGM_1的同步电泳分型及其在上海地区的分布与频率

用 pH7.4的 Tris-马来酸缓冲系统和混合淀粉凝胶同步检测血液及血癌中 EsD 和 PGM_1的表型,获得良好的分型效果。EsD 和 PGM_1的图谱区带平直、狭窄、清晰。各种表型之间差异著,极易区分容易发现稀有表型。我们在上海地区居民中检查了390人的 EsD 表型和724人的 PGM_1表型,其分布与其基因频率详见附表。在检测尸体血及尸体血痕时,发现一例尸体血和一例尸体血痕的 PGM 1活性明显增强,前者尚显现了一条额外的同工酶区带。     

PGM1 subtypes determined by agarose gel isoelectrofocusing

PGM1 subtypes were determined in red cell hemolysates by isoelectric focusing on agarose gel plates. By this modified procedure PGM1 subtypes may be readily classified. Nine of the 10 expected phenotypes were found in a sample of 470 unrelated individuals from Southern Germany. The frequencies for the four alleles were found to be: PGM1(1+) = 0.212, PGM1(1-) = 0.1224, PGM1(2+) = 0.2043, PGM1(2-) = 0.0521.     

Rare PGM1 (6) and PGM1(7) alleles in the Polish population

Rare PGM1 phenotypes, 6-1, 6-2 and 7-2, were detected in blood samples from 3,437 non-related adults using electrophoresis in starch-gel and cellulose acetate membranes. Frequencies of 0.0009 and 0.0008 were calculated for PGM1(6) and PGM1(7), respectively, for a population from northern Poland. The variants had been inherited, which was confirmed by family studies.     

Isoelectric focusing studies on the PGM1 subtypes in the northern Japanese population

The distribution of the human red cell phosphoglucomutase (PGM1) subtypes in samples from Japanese population (n = 277) living in the Miyagi Prefecture, the northern part of Japan, was investigated by applying the thinlayer polyacrylamide gel isoelectric focusing. In our population sample all the ten common phenotypes were demonstrated, and the estimated allele frequencies for the genes PGM1+1, PGM1-1, PGM2+1, and PGM2-1 were 0.671, 0.107, 0.161, and 0.061, respectively. Family studies (n = 40) indicated an autosomal codominant inheritance and confirmed the four alleles. The new system will increase the probability of exclusion in paternity cases among Japanese to 29.4% compared with 14.3% if the two allele system is used.     

Red cell phosphoglucomutase (PGM1) subtypes in Egyptians

The polymorphism of the human red cell phosphoglucomutase 1 (PGM1) in samples from Egyptians (n = 134) was investigated using isoelectric focusing in thin-layer polyacrylamide gel. In the studied population samples nine common phenotypes were observed, and the calculated frequencies for the genes PGM1+1, PGM1-1, PGM2+1 and PGM2-1 were 0.6381, 0.0821, 0.2201 and 0.0597, respectively. The observed and expected phenotypes provide a good fit to Hardy-Weinberg equilibrium. The four alleles system will increase the probability of excluding a man falsely accused of paternity to 30% as compared with 16% if the two alleles system is used.     

Phosphoglucomutase subtyping in a south Polish population

Phosphoglucomutase (PGM1) subtypes in South Polish population were examined by thin-layer polyacrylamide gel isoelectrofocusing (pH 5-7) using fresh hemolysates from 460 unrelated adults. The allele frequencies in Polish population are as follows: PGM1+1 = 0.6402, PGM1-1 = 0.1185, PGM2+1 = 0.1880, PGM2-1 = 0.0533.     

The examination of vaginally inserted plastic tampon applicators for genetic markers and evidence of prior sexual intercourse

Vaginally inserted plastic tampon applicators were obtained from 42 female volunteers. The applicators were examined for the presence of ABH blood group substances, phosphoglucomutase (PGM), amylase, acid phosphatase, P30, and intact spermatozoa. Each applicator was accompanied by a control blood sample, a saliva specimen, a brief sexual and menstrual history, and method of birth control of the donor. Eight of the male sexual partners of the donors submitted blood and saliva samples. One male sexual partner submitted only a saliva sample. ABH blood group substances corresponding to the donor were recovered from 36 of the 42 applicators. The remaining 6 applicators revealed a combination of the donor's and sexual partner's ABH substances. The female's PGM type was recovered from 34 of the applicators. The remaining 8 applicators failed to show PGM activity. Of the applicators, 15 indicated evidence of prior sexual intercourse by the detection of ABH substances not consistent with the applicator donor (6 samples), high levels of acid phosphatase (11 samples), or recovery of spermatozoa (8 samples) or some combination of these. All applicator samples failed to show the presence of either P30 activity or PGM factors foreign to the female.     

应用Ion Torrent PGM^TM平台进行人mtDNA全基因组测序

目的应用Ion Torrent PGM^TM平台对人线粒体基因组全序列进行分析检测。方法采集39名辽宁汉族无关个体以及4个母系家系的14名相关个体样本,应用SequalPrep^TM Long PCR试剂盒进行扩增,应用Ion Shear^TM Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM平台上进行线粒体基因组全序列测序。结果在39名无关个体共观察到39种单倍型,在396个位置观察到了397种碱基变异。无关个体中出现的变异位点数目为25~53个,平均每个个体出现36.2个碱基变异。4个母系家系中每个家系成员间具有完全相同的mtDNA单倍型,严格遵守母系遗传。结论采用本研究建立的人线粒体基因组全序列的测序检验法,可显著提高mtDNA的个体识别能力,在法庭科学领域中有较好的应用价值。