应用PCR-SSCP技术检测PGM1基因型

目的应用PCR-SSCP技术分型PGM1基因型.方法提取156份武汉地区汉族无关个体的血样DNA,分别扩增PGM1基因外显子4和外显子8的多态性靶DNA,用SSCP分析PCR产物,判断基因型.结果两种PCR产物均检出了两个等位基因、三种基因型,DP值分别为0.5620、0.4405.综合外显子4和8的PCR-SSCP结果,分出8种PGM 1基因型,DP值为0.731 8.应用本法对保存10年的陈旧血痕和精斑PGM1分型成功.结论用PCR-SSCP分型PGM1基因型在法医物证检验中具有实用价值.     

红细胞和血痕酸性磷酸酶(EAP)表型及广东人群EAP基因频率的调查

本文采用薄层聚丙烯酰胺凝胶等电聚焦电泳检测红细胞及血痕酸性磷酸酶表型,并对不同条件下的血痕标本进行检测,发现室温下(15～33℃)保存的110例纱布血痕7周内可全部正确分型,21例磁板血痕9周内均可正确分型;含血量≥5λl 的血痕可被正确检出 EAP 表型;日晒、水洗、发霉等因素可影响血痕 EAP 型的正确检出。同时调查了广东人群的 EAP 表型分布,基因频率为 p~a=0. 2338,p~b=0. 7662,发现 EAP 基因频率分布存在着地区差异。     

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     

基于高通量测序进行无创产前亲子鉴定的可行性

目的初步探讨基于高通量测序进行STR分型的技术方法应用于无创产前亲子鉴定的可行性。方法选择13个STR基因座(6个常染色体STR基因座,6个Y染色体STR基因座,1个性别判定基因座),进行复合PCR扩增和高通量测序文库构建后,采用Ion PGM400高通量测序平台进行测序,并采用自主研发软件NGS-STR genotyper(perl脚本)进行STR分型,本文简称上述过程为NGS-STR分型。对13个母子配对混合样本(母亲:儿子=2%~50%)、1组家系样本进行了上述NGS-STR分型,旨在(1)了解其在混合样本中的灵敏度及分型情况;(2)了解其在无创产前亲子鉴定中的应用可能性。结果 (1)当混合样本中低组分(儿子)的比例超过8%,所有基因座均可检出低组分的STR信息;(2)对1例血浆样本进行NGS-STR分型,共计69.2%的基因座可检出胎儿的STR基因型信息,且所有检出基因座均符合孟德尔遗传规律。结论初步证明了NGS-STR分型技术具有进行无创产前亲子鉴定的可行性。     

成都地区汉族Gc亚型的分布及血痕中Gc亚型的检测

作者用免疫固定薄层聚丙烯酰胺凝胶等电聚焦(PAGIF)技术,调查了成都地区无关的125名健康汉族人血清Gc亚型分布。其6种亚型频率(%)分别为:Ge1F=20.8,Ge1S=8.0,Gc1F-1S=18.4,Gc2-1F=30.4,Gc2-1S=16.0和Gc2=6.4。Gc的基因频率为:Cc~(1F)=0.452,Gc~(1S)=0.252和Gc~2=0.296。对保存于室温条件下20周的陈旧血痕进行了Gc亚型定型,获得满意结果。     

红细胞酸性磷酸酶(EAP)型的分布及血痕EAP的检出

本文应用琼脂糖凝胶电泳法对辽宁地区213例汉族随机献血员的红细胞酸性磷酸酶(EAP)型进行了检测,其基因频率为EAP~(?)=0.169,EAP~b-0.831。结合文献资料分析了EAP型分布的种族差异,指出了各人种EAP型分布的特点。用琼脂糖凝胶电泳法,对红细胞溶血液EAP型的最小检出量为2μL(2×3mm滤纸条)及0.5μL(直接加样);对血痕EAP型的最小检出量为7.5μL血液制成的检样。本法在4℃保存4周以内的红细胞溶血液和室温保存25天的血痕均能正确判定EAP的型别。     

中国人血清脱氧核糖核酸酶I(DNase I)的遗传多态性研究

应用薄层PAGIF（T=5％，C=3％）结合特异酶底物染色技术，调查了中国随机人群DNaseI遗传多态性的分布，检出在中国人群中两种常见的等位基因，即DNaseI*1和DNasel*2，其基因频率DNaseI*1为0．53，DNaseI*2为0．47。家系分析表明：子代个体谱带分别来自父亲和母亲，谱带在亲代和子代之间的传递符合孟德尔遗传规律。按Hardy－Weinberg法则进行吻合度检验，观察值与期望值一致。人血清DNaseI等电点经测定为4．0。     

血痕中PGM_1亚型的检出及酶谱的保存方法

本文报告用等电聚焦方法,采用拉丁方设计,对血痕保存的温度、布质及含量,以及血痕中 PGM_1亚型检出时间进行了研究。保存在0℃(6个月)、4℃(2个月)、18℃(1个月)及30℃(3周)的6μL 血痕,PGM_1亚型均可检出。血痕的总量对 PGM_1亚型的检出时间也有一定的影响。不同布质对血痕中 PGM_1亚型的检出时间,无明显差异。另外,利用聚脂膜具有亲水膜面的特点,将 PGM_1原始酶谱贴附在聚酯膜上,可长期保存酶谱。     

签字笔上附着的脱落上皮细胞STR分型

目的 探讨遗留在签字笔上微量脱落细胞DNA分型的可行性以及保存时间对分型的影响.方法 17名志愿者每人使用7支签字笔,每支笔每天使用20 min,为期1个月,分别保存1、3、5、7、14、21和28 d,运用硅珠法提取签字笔上微量脱落细胞中的DNA,应用荧光标记PCR-STR技术进行DNA分型,同时采集上述17名志愿者口腔拭子作为对照,分析签字笔作为检材进行DNA分型的可行性以及保存时间对DNA分型的影响. 结果以基因座检出个数为指标,签字笔脱落细胞和口腔拭子的DNA分型结果随保存时间变化而产生的差异具有统计学意义(P＜0.01).签字笔保存1、3、5、7、14、21和28 d后进行DNA分型检出的基因座个数与对应的口腔拭子DNA分型检出的基因座个数相比差异均有统计学意义(P＜0.01).签字笔使用后保存1 d进行DNA分型.可明确判读12个以上基因座的占41.2%. 结论签字笔上附着的微量手指脱落细胞可作为一种法庭生物检材进行DNA分型,但其保存时间会影响DNA分型.     

精斑中磷酸葡萄糖变位酶(PGM_1)及其亚型的电泳分型

本文用淀粉凝胶电泳法和 PAGIEF 对精斑 PGM_1普通型及亚型进行了检测。169份精液斑的 PGM_1分型结果是:PGM_1 1—1 87例;PGM_1 2—1 66例;PGM_1 2—2 16例,其中31例同一个体红细胞及精液 PGM_1分型的结果完全一致。研究了138例不同精子数精斑的 PGM_1型,发现精子数的多少对分型无影响。亚型检测结果与红细胞一样可分10型。     

Simultaneous phenotyping of EAP and PGM1 by PAG1F

The study is to find new genetic markers of simultaneous phenotyping and to raise the determinating power of isoenzymes. Simultaneous electrophoresis of EAP and PGM1 by thin-layer PAG1F combined with reverse zymogram developing was applied and clear isoenzyme bands was observed on both surface of a gel without interference. The method can do typing of the EAP and PGM1 at one gel and the accumulative DP is 0.87. It can be used in paternity test.     

Studies on the use of isoelectric focusing as a method of phenotyping erythrocyte acid phosphatase

The technique of isoelectric focusing (IEF) in ultra-thin polyacrylamide gels as a method of phenotyping erythrocyte acid phosphatase (EAP) has been applied to a large number of red cell lysates and dried bloodstains. This paper presents the results of this study and discusses some features of the IEF patterns and problems with their interpretation. The IEF patterns of several rare EAP phenotypes are also described. These studies have confirmed that IEF is more sensitive than starch gel electrophoresis as a method of phenotyping EAP in dried bloodstains.     

Usefulness of two methods of isoelectric focusing for the erythrocyte acid phosphatase phenotypes determination in human bloodstains

The authors tried to compare the usefulness of the isoelectric focusing of EAP in bloodstains on 0.2 mm polyacrylamide gel with their method of determination of the enzyme on 1 mm polyacrylamide gel. Both methods turned out to be useful but better results were obtained on 0.2 mm gel. Isoelectric focusing on the ultra-thin gel is more sensitive; it gives clear enzyme strips, takes less time (30 min) and demands about half the amount of material.     

Bloodstain characterization in the EAP, Hp, Hb, AK and Glo I typing systems using minigels and the PhastSystem

Application of minigels and the PhastSystem to obtain phenotyping results from bloodstains in the EAP, Hp, AK, and Glo I typing systems was investigated. Nonequilibrium isoelectric focusing with 4-6.5 PhastGel produced readily interpretable phenotypes in the EAP typing system. Both 4-6.5 and 5-8 PhastGel produced AK typing system phenotypes using nonequilibrium isoelectric focusing conditions. The 8-25% PAG PhastGel developed by two staining techniques allowed discrimination of phenotypes for the Hp typing system. Phenotypes from the Glo I typing system were also obtained with this gel type. Variant haemoglobins could be detected on pH 5-8 PhastGel using isoelectric focusing conditions. Much potential for standardized, rapid phenotyping of bloodstains was found to exist utilizing the PhastSystem.     

The use of separator isoelectric focusing in micro-ultrathin polyacrylamide gels in the characterization of some polymorphic proteins of forensic science significance

The identification of phenotypes of erythrocyte acid phosphatase (EAP), esterase D (EsD), group specific component (Gc), and alpha-1-antitrypsin (PI) by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 45 mm) is described. The protein patterns obtained are compared favorably with the patterns seen by isoelectric focusing in conventional polyacrylamide gel dimensions (interelectrode distance: 110 to 120 mm). The technique described allows greater stability of pH gradients and is a fast and economic method.     

The use of ultrathin-layer agarose gels for phenotyping erythrocyte acid phosphatase by isoelectric focusing

A method is described for the use of ultrathin-layer agarose gels in phenotyping erythrocyte acid phosphatase (EAP) by isoelectric focusing (IEF). The results obtained using ultrathin-layer agarose gels are shown to be equally reliable and reproducible in comparison to established ultrathin-layer polyacrylamide gels. IEF of EAP on 0.168-mm agarose gels took place in 90 min using the LKB Multiphor system. The technique described allows for both time and cost efficient phenotyping of EAP.     

Simultaneous electrophoretic determination of phosphoglucomutase subtypes, adenosine deaminase, erythrocyte acid phosphatase, and adenylate kinase enzyme phenotypes

Many of the conventional agarose phosphoglucomutase (PGM) subtyping systems presently in use fail to provide a good separation between the 1 + and 2- bands as well as the 2+ band and the more anodic moving bands. Use of a 1-mm-thick gel composed of 1% ISO GEL (FMC Corp.) and phosphate-citric acid gel and tank buffers with a pH of 5.3 provided exceptionally good separation between all four of the major subtyping bands. The additional criteria for this procedure is a voltage of 21 V/cm and a run time of 4 h. Utilization of this procedure using case samples of varied ages proved the reliability of the procedure. Also examined were the effects of several reducing agents on the enzyme band patterns and the use of this system for the simultaneous determinations of the adenosine deaminase (ADA), erythrocyte acid phosphatase (EAP), and adenylate kinase (AK) enzyme phenotypes.     

The use of isoelectric focusing (IEF) as a method for the combined phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (EsD)

The simultaneous isoelectric focusing (IEF) in polyacrylamide gels (PAG) of erythrocyte acid phosphatase (EAP) and esterase D (EsD) allows the poor discriminating power (DP) of EsD to be usefully combined with a highly discriminating system EAP, such that a joint DP of 0.766 was achieved compared with PGM IEF DP 0.756. Focusing was carried out in a centrally flattened gradient containing ampholines (pH 4-6 and 6-8) and the chemical spacer 3-(N-morpholino) propanesulphinic acid (MOPS). It enabled the identification of six EsD phenotypes including the recently discovered EsD5 isozymes. The application of this method to casework bloodstains is discussed.