数字编码小卫星可变重复序列法对中国汉人分型

介绍用辣根过氧化物酶标记的寡核苷酸探针检测的数字编码小卫星MS32可变重复序列方法。经用此法研究发现，中国汉人可得到50个编码以上，无关个体前50个编码中至少有20个编码不同，未发现两个无关个体的编码相同，50个编码全部相同的概率为4．09×10-16。三种重单位，即a-型、t-型和o-型出现比例分别为61．5％、32％和6．5％。MVR－PCR得到的数字编码具有高度的变异性，识别率高，为法医物证检验提供一个新的个人识别方法。     

中国朝鲜族线粒体DNA编码区序列多态性

目的调查中国朝鲜族群体线粒体DNA（mtDNA）编码区内5个部位 3954～4506nt、5218～5974nt、7942～871Int、10296～10653nt 及14496～14867nt的序列多态性。方法采用PCR产物直接测序方法,对212名中国朝鲜族（吉林省延边地区）无关个体进行序列多态性变化和单倍型分布调查。结果在212名无关个体中,共分出148种单倍型。遗传变异度为0．9931,耦合概率为0．0116。测序结果与Anderson标准序列比较,共检测出109个变异位点,其中79个已收录于MITOMAP。结论mtDNA编码区多态性联合应用可以提高mtDNA的个体识别能力。町为相关遗传学研究提供基础数据资料。     

MS31A的MVR-PCR分型

建立一种快速MVR－PCR分析方法,对中国汉族群体样本进行小卫星MS31A（D7S21基因座）分型及其频率调查。采用同位素掺入扩增和中性聚丙烯酸胺凝胶电泳分离技术。对200名无关个体血液样品的分析结果表明,每一样品MVR－PCR图谱均可获得近40条谱带。经计算机编码分析,不同个体的编码均不相同。MS31A前40个重复单位1、2、3、4型的频率分别为25．3％,27．2％,47．3％和0．2％;DP值可达99．999999999999％;四代家系分析表明：亲代小卫星MS31A的重复单位可稳定地传递给子代,符合孟德尔遗传规律。灵敏度分析显示：该技术最低能检测出1ng的基因组DNA.MW－PCR分析方法在法医学个人识别方面具有应用价值。     

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。     

应用Ion Torrent PGM?平台进行人mtDNA全基因组测序

目的应用Ion Torrent PGM?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SequalPrepTMLong PCR试剂盒进行扩增,应用Ion ShearTMPlus 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。     

线粒体DNA编码区的多态性

目的研究线粒体DNA(mtDNA)编码区单核苷酸多态性,建立mtDNA编码区多态性在法庭科学中应用的理论基础。方法针对mtDNA编码区nt8162-8483以及nt13070-13299两段序列设计引物,应用直接测序技术研究其多态性。结果两对引物扩增片段长分别为322bp和230bp,共检测到21种变异,24种单倍型,基因多样性为0.7511,两个无关个体的偶合概率为0.2564。结论线粒体DNA编码区多态性位点作为线粒体DNA控制区多态性位点的补充,联合应用可以提高线粒体DNA在法医学应用中的个体识别能力。     

中国广东汉族群体mtDNA控制区的多态性

目的 探讨线粒体DNA控制区(包括HVⅠ区、HVⅡ区和HVⅢ区)的多态性。方法 采用PCR扩增和末端标记荧光循环测序的方法，对100名广东汉族无关个体进行了序列分析。结果 共观察到147个变异位点，序列变异包括了碱基转换、颠换、插入、缺失等各种类型。其中在HV Ⅰ区(nt16，024～nt16，365)内观察到88个变异位点，91种单倍型，基因多样度为0.9964；在HVⅡ区(nt73～nt340)观察到42个变异位点，67种单倍型，基因多样度为0.9861；在HVⅢ区(nt438～nt574)观察到9个变异位点，15种单倍型，基因多样度为0.8760。联合3个高变区域的序列，可观察到98种单倍型，基因多样度为0.9996。结论 本研究为线粒体DNA在法庭科学中的应用提供了较系统的实验依据。结果还表明，对于mtDNA等单倍型遗传标记，增加其检测范围，可提高该系统的个体识别能力，使其在法庭科学领域充分发挥作用。     

中国汉族人群的线粒体DNA控制区多态性研究

探讨mtDNA多态性在法庭科学中个体识别的理论基础。应用PCR扩增产物直接测序方法 ,对 111名中国北方地区汉族人群无血缘关系个体的mtDNA控制区 (HVⅠ和HVⅡ )进行测序分析。在高变区Ⅰ 15 998～ 16 40 0之间发现 10 2处碱基变异 ,10 3个mtDNA单倍型 ;在高变区Ⅱ 0 0 0 35～ 0 0 36 9之间的发现 36处碱基变异 ,6 9个mtDNA单倍型。其可变碱基的变异形式主要为碱基替代 (转换和颠换 )、插入和缺失 ;碱基转换 (78 9% )明显高于颠换(14 3% )、插入 (3 4% ) ,缺失 (3 4% )。分析表明 ,人群个体mtDNA控制区碱基序列 ,基因多样性为 99 9% ,两个无关个体的偶合概率为 0 92 % ,具有高度序列的多态性     

用dHPLC技术检测线粒体DNA编码区单核苷酸多态性

目的研究线粒体DNA(m tDNA)编码区单核苷酸多态性,建立检测m tDNA编码区单核苷酸多态性(SNP)的变性高效液相色谱(dHPLC)方法。方法设计针对线粒体DNA编码区nt10287-10679及nt8507-8805引物,应用dHPLC技术检测其序列多态性。结果100例中国汉族无关个体中,m tDNA nt10287-10679检出13个SNP位点,13种单倍型,基因多样性(H)为70.79%,偶合概率(P)为29.92%;m tDNA nt8507-8805检出10个SNP位点,12种单倍型,H为70.42%,P为30.28%;两段序列联合起来共检出23个SNP位点,23种单倍型,H为84.14%,P为16.70%。结论所建立的dHPLC方法可用于快速、准确地检测m tDNA编码区序列多态性;m tDNA编码区多态性位点作为m tDNA控制区多态性位点的补充,联合应用可以提高m tDNA的个体识别能力。     

中国北方汉族人群HLA-DQα基因型及其等位基因频率的分布

应用PCR和等位基因特异性寡该苷酸（ASO）探针杂交技术对中国北方汉族人群的HLA－DQα基因进行分型。在246例无关个体中观察到4种等位基因组成的10种基因型。等位基因频率分布在10．0～31．9％之间。基因型的分布符合Hardy－Weinberg定律。DP值为0．8669。对6个家系49个个体的调查表明，HLA－DQα基因按孟德尔方式遗传。不同人群HLA－DQα的DP值比较，中国人群高于其它人群。     

Y-chromosome variation in a Norwegian population sample

Y-chromosome DNA profiles are promising tools in population genetics and forensic science. Here we present DNA profiles of 300 unrelated Y-chromosomes of Norwegian origin. The profile is composed of eight short tandem repeats (STRs) and one single nucleotide polymorphism (SNP). In more than 2/3 of the haplotypes the modular structure in the 5' end of the minisatellite locus DYF155S1 was revealed by minisatellite variant repeat PCR (MVR-PCR) These haplotypes were also typed for deletions of fragment 50f2C (DYF155S2). Allele distribution and paternity exclusion parameters are given for each marker. The degree of haplotype diversity and its implication for statistics are evaluated. In the 300 samples 177 different haplotypes were encountered, of which 137 were observed once only. Analysis showed that the main source of variation is within the population. The Fst values were less than 0.015 in general. Haplotype grouping by the SNP demonstrated two haplogroups (Tat/T and Tat/C). Haplogroup Tat/C--found in 5.7% of the present material - is the same haplogroup as encountered in 60% of Finnish males [Am. J. Hum. Genet. 62 (1998) 1171]. Mutation analysis in 150 father/son pairs (a total of 1200 meiotic events) revealed an average mutation frequency of 0.0042 (95% CI 0.0014-0.0097).     

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.     

中国人p33.6位点的扩增片段长度多态性

用PCR、小型聚丙烯酰胺凝胶电泳和银染法对小卫星区域p33.6(D1S111)位点的扩增片段长度多态性(Amp—FLP)进行分析和对100例无关中国人p33.6位点的等位基因频率进行调查及数据处理,发现该位点核心序列重复数从9到22之间的全部14个等位基因,片段长度分布于435～925bp之间,基因频率为0.5～35.5％,杂合度为76％。对6个家系共22名相关个体进行分析,符合孟德尔遗传定律;对人体各种不同组织DNA进行该位点的分析,显示出高度的一致性。该位点适用于法医学上的个人识别以及亲子鉴定。     

Distinguishing minisatellite mutation from non-paternity by MVR-PCR

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was devised to map the interspersion pattern of subtle variant repeats along minisatellite tandem arrays. MVR-PCR has revealed enormous diversity of allele structures at several loci, far more than can be resolved by allele length analysis. We have reported the application of MVR-PCR at minisatellite MS32 (D1S8) and MS31A (D7S21) in a paternity case lacking a mother and showed that it resulted in higher paternity probabilities than for a set of 12 other DNA markers including six STRs. Hypervariable minisatellites like MS32 and MS3lA can however, show significant germline mutation rates to new length alleles which can generate false exclusions in paternity cases although paternity cases showing mutant paternal alleles at more than one locus will be rare when several MVR loci are examined. Detailed knowledge of mutation processes coupled with MVR analysis of allele structure can help distinguish mutation from non-paternity. We now show how similar mutant alleles are to their progenitors using both real and simulated data, and demonstrate how MVR-PCR can be used to identify mutant paternal allele in paternity cases showing apparent exclusions.     

Highly polymorphic minisatellite DNA probes. Further evaluation for individual identification and paternity testing

Reliable and reproducible protocols have been developed for the routine DNA fingerprinting of individuals using the highly polymorphic minisatellite DNA probes 33.15 and 33.6. Comparison of DNA fingerprinting from 50 individuals has generated further data on the level of band sharing in the DNA fingerprints of unrelated individuals, as well as the number of bands scorable in individuals. These results are consistent with previous studies. The occurrence of mutant bands in offspring has been examined in over 100 families. Further support is presented for the Mendelian inheritance of minisatellite loci and for lack of significant allelism and linkage between different variable DNA fragments detected in a human DNA fingerprint.     

中国汉族与日本群体DYFl55S1基因座的遗传多态性

目的探讨Y染色体DYF155S1基因座的遗传多态性及群体间差异。方法 应用MVR-PCR、荧光显谱及DNA序列分析技术,对来自中国群体(北方汉族,64例)和日本群体(43例)男性个体的DYF155S1基因座进行初步分析。结果107例样本共检出了5种重复序列类型,包括新的命名为6型的重复序列,它是在1型的基础上T22A置换所形成,仅存在于日本群体,可作为民族特征性遗传标记。2群体重复序列的排列方式以3134顺序为主,在中国和日本群体中各占73.44％和67.44％,是黄种人的特点。134顺序在中国群体中占第二位,为17.19％,6134排列占日本群体的16.28％。3’端的4型重复序列的平均数目在日本群体为8.8条,明显低于中国群体的12.5条。结论DYF155S1基因座具有非常高的遗传多态性和明显的群体差异。     

Genetic variation and population genetic data of the short tandem repeat locus D8S320

The locus D8S320 is an STR system first described in 1993 as a simple (AAAG) repeat. Sequencing data revealed that the D8S320 locus is a complex STR system consisting of (AAAG)- and (AAAC)-repeat units. A total of 22 different alleles were found in a population survey of 210 unrelated individuals from the Rhine area with frequencies ranging from <0.01 to 0.198. The population data revealed the existence of variant alleles differing by 1 and 2bp from the consensus allele. Due to the complex repeat structure consisting of three variable regions and one constant region, electrophoresis under denaturing conditions is strongly recommended. The statistical values were calculated to be 0.89 (observed heterozygosity rate), 0.96 (discrimination index) and 0.71 (mean exclusion chance). No deviation from Hardy-Weinberg equilibrium (HWE) was observed.     

Identification and characterization of variant alleles at CODIS STR loci

Short tandem repeat (STR) profiles from 32,671 individuals generated by the ABI Profiler Plus and Cofiler systems were screened for variant alleles not represented within manufacturer-provided allelic ladders. A total of 85 distinct variants were identified at 12 of the 13 CODIS loci, most of which involve a truncated tetranucleotide repeat unit. Twelve novel alleles, identified at D3S1358, FGA, D18S51, D5S818, D7S820 and TPOX, were confirmed by nucleotide sequence analysis and include both insertions and deletions involving the repeat units themselves as well as DNA flanking the repeat regions. Population genetic data were collected for all variants and frequencies range from 0.0003 (many single observations) to 0.0042 (D7S820 '10.3' in North American Hispanics). In total, the variant alleles identified in this study are carried by 1.6% of the estimated 1 million individuals tested annually in the U.S. for the purposes of parentage resolution. A paternity case involving a recombination event of paternal origin is presented and demonstrates how variant alleles can significantly strengthen the genetic evidence in troublesome cases. In such instances, increased costs and turnaround time associated with additional testing may be eliminated.     

The application of minisatellite variant repeat mapping by PCR (MVR-PCR) in a paternity case showing false exclusion due to STR mutation

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged father's medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boy's allele 17 could have originated from either of the alleged father's allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.