PCR-based DNA analysis of vWA31A,TH01, F13A01, FESFPS,TPOX and CSF1PO

The allele and genotype frequencies of 6 tetranucleotide STR loci were investigated in a sample of 132 unrelated individuals from Chinese Han population. The PCR products were analyzed on 6% denaturing PAGE and detected using fluorescently labeled primers in an automated 377 sequencer(PE). All loci meet Hardy-Weinberg equilibrium. There was no random association of alleles among the 6 loci. The allele frequencies were compared with other population databases. Except locus vWA31A, the observed heterozygosity at other 5 loci was significantly lower than that reported in Caucasian and Black population studies. The calculated DP = 0.99999, PE = 0.9708, pM = 1.059 x 10(-5). The allelic frequency data can be used in forensic identification and paternity testing.     

A novel fluorescent quadruplex STR typing system and the allele frequency distributions in a Thai population

We have previously reported a new triplex amplification and typing system by silver staining for three short tandem repeat (STR) loci, 9q2h2 (D2S3020), D15S233, and D14S299 without "microvariant" alleles such as .1, .2, and, .3 alleles in the Japanese population. In the present study, we established a new quadruplex system with an additional locus D7S809 using primer sets labeled with fluorescent multi-color dyes. Using this system, we genotyped 183 Thai people, found only one "microvariant" allele (allele 20.2) at D7S809, and calculated allele frequencies and some statistical properties at these four STR loci. From these allele frequencies at four STR loci, we performed three statistical analyses including a homozygosity test, a likelihood ratio test, and an exact test for Hardy-Weinberg equilibrium (HWE). Deviations from HWE (p < 0.05) were observed only in the two tests at the locus D7S809. In the present study, we compared the allele frequencies at these four loci in the Thai population to those in the Japanese population described previously. Consequently, all observed heterozygosities and power of discrimination (PD) at those loci in the Thai population were higher than 0.8 and 0.9, respectively, and all statistical values for discriminating power in the Thai population were slightly higher than those in the Japanese population. The combined paternity exclusion rate (combined PE) in the Thai population (0.978) was almost the same as that in the Japanese population (0.971). Therefore, this novel PCR amplification and typing system for four STR loci would be a convenient and informative DNA profiling system in the forensic field.     

Updated Brazilian genetic data, together with mutation rates, on 19 STR loci, including D10S1237

Allelic frequencies for 19 STR loci (F13B, TPOX, D3S1358, FGA, CSF1PO, D5S818, F13A01, D7S820, D8S1179, D10S1237, TH01, VWA, D13S317, FESFPS, Penta E, D16S539, D18S51, D19S253, and D21S11) were obtained from an average of 13,000 unrelated Brazilian adults undergoing parentage testing. D10S1237 is a tetranucleotide repeat locus shown to be useful for forensic and paternity studies. Null allele frequencies and mutation rates were ascertained from this population sample.     

Identifiler^TM系统在亲子鉴定中的突变观察和分析

目的观察和分析IdentifilerTM系统15个短串联重复序列(STR)位点在亲子鉴定中的突变现象。方法用IdentifilerTM试剂盒检测2712例亲子鉴定案例。结果在2362例认定亲子关系的案例中,观察到51例中有1个STR位点发生突变。突变的位点包括D8S1179、D21S11、D7S820、CSF1PO、D3S1358、D13S317、D16S539、D2S1338、D19S433、vWA、D18S51、D5S818和FGA。其中以D21S11位点突变率最高(0.369%);突变的等位基因来自父亲36次,来自母亲7次,无法确定9次。结论STR位点突变是较为常见的现象,采用IdentifilerTM系统进行亲子鉴定,遇到1～2个STR位点不符合遗传规律时,有必要增加突变率低、稳定性好的STR位点进行复核。     

Mutations of short tandem repeat loci in Identifiler system

OBJECTIVE: To explore and analyze the mutations of 15 Short Tandem Repeat (STR) loci using Identifiler system in paternity identification. METHODS: 2712 cases of paternity testing were carried out using Identifiler PCR Amplification Kit. RESULTS: Of the 2362 paternity testing cases, mutations of single locus were observed in 51 cases. The mutation loci included D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, D2S1338, D19S433, vWA, D18S51, D5S818 and FGA, with the D21S11 locus having a highest mutation rate (0.369%). Thirty-six of the STR mutations were from paternal source, 7 from maternal source, and the rest (9) were undeterminable. The mutation rates at D21S11 were highest (0.369%). CONCLUSION: Mutations of STR loci are relatively common in human genome. Therefore, retesting of additional relatively stable STR loci with lower mutation rates is necessary when one or two loci exclusions are encountered in paternity testing.     

D3S1754、D18S535基因座在中国北方汉族的多态性分析

目的 了解D3S1754、D18S535基因座多态性在中国北方人群中的分布特点及其应用价值。方法 使用PCR、聚丙烯酰胺垂直板电泳及银染的方法。结果D3S1754基因座检出9个等位基因（n=184）,D185535基因座检出8个等位基因（n=201）,两个位点的等位基因频率在群体中的分布符合Hardy-Weinberg平衡（P>0.05）,它们的杂合率（He）分别为0.706和0.807,个人识别机率（DP）分别是0.859和0.934,非父排除率（EPP）分别为0.464和0.629。结论 D3S1754、D18S535两个遗传标记的个人识别率高、非父排除能力较强且能稳定遗传,具有较高的应用价值。     

13个STR基因座在亲子鉴定案例中的基因突变观察

目的 观察美国 CODIS系统的 13个 STR基因座在532例认定亲子关系的亲子鉴定案中的基因突变情况,探讨STR基因座突变率及突变类型。方法 经“Profiler Plus”及“Cofiler”试剂盒检测的587例亲子鉴定案,对其中有1～2个STR基因座不符合遗传规律者,增加HLA等血型基因和“PowerPlex16~(TM)”试剂盒检测。必要时,还增加Y-STR基因座检测和HLA等位基因测序。结果 认定亲子关系的532例,观察1052次减数分裂,发现17例亲子鉴定中的18次基因突变事件,其中16例1个STR基因座的基因发生突变,1例2个STR基因座的基因发生突变;突变的基因座包括D5S818、D3S1358、D16S539、CSFIPO、D21S11、D13S317、D7S820、vWA、D18S51和FGA,其中以FGA和D18S51基因座的突变率最高(0.29％);18次突变事件,其中来自父亲11次,来自母亲5次,无法确定2次。结论 用美国CODIS系统的STR基因座进行亲子鉴定,在有1～2个基因座不符合遗传规律时,要综合分析,并增加其它的遗传标记进行检测。     

北京地区汉族人群6个STR基因座的遗传多态性

目的对北京地区汉族人群遗传数据进行调查研究。方法利用PCR自动化检测技术检测中国北京地区汉族人群D1S1656,D10S2325,D11S2368,D12S391,D14S1434及GATA198B05共6个STR基因座的遗传多态性,获得6个STR基因座的群体遗传学数据,评价其法医学应用价值。结果6个STR基因座的个体鉴别力（Discrimination power,DP）在0．9777-0．8769之间,多态性信息含量（Polymorphism information content,PIC）在0．6867-0．8801之间,杂合度（Heterozygosity,H）在0．7219～O．8684之间,非父排除率（Probability of paternity exclusion,PE）在0．4456-0．6793之间,累积个体鉴别力为0．99999997,累积非父排除率为0．995765192。结论6个STR基因座等位基因频率分布均匀,多态性高,适用于法医学亲子鉴定及个人识别,可作为现有基因座的补充。     

D6S1043和D12S391基因座在亲权鉴定中的应用

目的研究D6S1043和D12S391基因座在亲权关系鉴定案件中的应用价值。方法应用荧光标记复合扩增系统对日常检案中所收集的192名汉族无关个体血样DNA进行PCR扩增,用ABI3100-Avant遗传分析仪对扩增产物进行毛细管电泳,用GeneMapperv3.2软件进行基因分型,统计分析D6S1043和D12S391基因座的多态信息。结果在D6S1043和D12S391基因座分别发现12个等位基因,它们在中国汉族人群中的个体识别能力分别为0.9656和0.9510,二联体非父排除率分别为0.573和0.510,三联体非父排除率分别为0.731和0.679。结论D6S1043和D12S391基因座具有高度多态性,在亲权鉴定中具有重要应用价值。     

Frequencies of D8S384 alleles and genotypes in European, African-American, Chinese, and Japanese populations

D8S384 is a tetranucleotide tandem repeat locus. In order to evaluate the forensic validation of D8S384, the genotype distributions and allele frequencies in ten populations from three main ethnic groups were investigated, including Germans, Slovakians, African Americans, Japanese, and Chinese (Jilin, Guangzhou, Nanning, Hailaer, Dali, and Chengdu). A total of 1011 unrelated individuals, 41 pedigrees, 30 disputed paternity trios and three personal identification cases were analyzed for D8S384 by Amp-FLP technique. Many kinds of tissues, body fluids, secreta and stains have been tested. The alleles were determined by comparison with a human allele ladder. The results showed that D8S384 typing was both precise and reliable. There were eight alleles in these populations. The genotype distributions conformed to Hardy-Weinberg equilibrium predictions. No mutation events were observed. With a maximum likelihood method, the mutation rate was indirectly estimated as 2.14 x 10(-5). The heterozygosity was 0.704 +/- 0.014 at D8S384 locus. All these results suggest that D8S384 locus is a useful marker for forensic identification and paternity analysis.     

北京汉族群体9个STR位点的频率分布及法医学应用

提供北京汉族群体9个STR基因座的频率分布资料，了解其在法医学中的应用价值。应用PCR技术对9个STR基因座分3组进行复合扩增，经PAG电泳分离、银染，扫描仪扫描，计算机判读并保存结果，对北京地区汉族无关个体9个基因座的基因频率分布进行调查。结果显示，上述9个基因座的杂合度为0．6419～0．8092，多态性信息总量为0．9999，鉴别机率为0．9999，匹配机率为2．0×10-9和非父排除率为0．9985。STR3组9个基因座的综合检验可应用于法医学个体识别和亲子鉴定，并达到同一认定的标准。     

CODIS位点在排除亲权中的应用价值

目的 对 CODIS位点 (FGA、 vWA、 CSF1PO、 TH01、 TPOX、 D3S1358、 D5S818、 D7S820、 D8S1179、 D13S317、 D16S539、 D18S51和 D21S11共 13个 STR位点 )在 100例排除亲权的亲子鉴定中的应用价值进行研究。方法 采用 Profiler Plus及 Cofiler荧光标记复合扩增系统,通过 310遗传分析仪对上述二个检测体系扩增产物的基因型进行分析。结果 在排除亲权的母亲－孩子－假设父亲三联体组中,所有观察案例其出现的排除指标数都在 3个以上,平均排除指标数为 6.63个;在假设父亲－孩子二联体组中, 94.0％的观察案例其排除指标数均在 3个以上,平均排除指标数为 5.01个。结论 CODIS位点在排除亲权的二联体和三联体组合亲子鉴定中,都符合鉴定应用要求 ;选择多态性较高的位点与增加排除指标存在直接的联系,以 DP、 H、 PE作为衡量 DNA位点应用价值的指标在具体鉴定实践中是可靠和可行的。     

Population genetic studies on the tetrameric short tandem repeat loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Egypt.

The short tandem repeat loci (STRs) D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 and a locus allowing for sex-discrimination (amelogenin) can be co-amplified by the polymerase chain reaction using a commercially available kit (AmpFlSTR Profiler plus, Perkin-Elmer Biosystems, San Jose, CA) and subsequently typed using capillary electrophoresis (ABI Prism 310 Genetic analyzer, Perkin Elmer Applied Biosystems, San Jose CA). To establish databases for these loci for an Arab population sample from Egypt, 140 unrelated persons were typed. Analysis of these data revealed that all loci except for VWA were in Hardy-Weinberg equilibrium, that the combined mean paternity exclusion chance (MEC) was 0.999875 and that the combined discriminating power (DP) was 2.635 x 10(-11). The allelic distributions found in the Egyptian sample were significantly different at four loci from those found for an Austrian Caucasian population, at all nine loci from an African-American sample and at six of six loci from a Chinese sample. No evidence of linkage equilibrium between any of the co-amplified loci was found. Our results support that the combination of multiplex PCR and capillary electrophoresis can both save time and yield excellent results for paternity testing and stain analysis.     

Analysis of human specificity in AFLP systems APOB,PAH, and D1S80

We have previously characterized and databased three human amplified fragment length polymorphism (AFLP) loci: the hypervariable regions 3′ to apolipoprotein B (APOB), phenylalanine hydroxylase (PAH) and at locus D1S80. The analysis utilized polymerase chain reaction (PCR) technology for human identification in forensic and paternity testing. This study extended that work by assessment of specificity of amplicons produced with non-human and human control DMAs for APOB, PAH and D1S80 under high and low stringency PCR conditions. It was seen that primate and other animal templates (with the exception of chimpanzee) yielded products below the human allele range under high stringency PCR parameters. Under reduced stringency PCR with animal and primate samples, reproducible genetic fingerprints were generated spanning the human allele range. The patterns were produced with defined human AFLP primer pairs under specifically relaxed PCR reaction and thermalcycling parameters. They showed genetic relationships between species at the DNA level. Amplicon patterns were compared for band size and intensity matches within the PCR synthesis range defined by the conditions used. This technique could become a useful tool in species identification and molecular evolutionary studies.     

D19S400基因座在中国3个汉族群体与日本人中的遗传多态性研究

为了解中国广州、吉林、成都三个地区汉族和日本人群体D19540O基因座基因频率分布，并获得中国三个汉族群体和日本群体D19M00基因座的群体遗传数据，比较它们之间的遗传学差异，探究在法医学应用中的意义。应用PCR扩增技术，聚丙烯酸胺凝胶垂直板电泳对D19S400基因座分型。在四个群体469个个体中共检出11个等位基因，45种基因型，基因型频率分布均符合Hardy-Weinberg平衡。各群体的观察杂合度为0.75～0.84，非父排除概率为0.6057～0.6582，个人识别机率为0．9301～0．9480。四个群体之间基因频率分布无显著性差异（P＞0．05）。结果表明，D19S400基因座在群体遗传学研究和法医学个人识别中有较高应用价值。     

短串联重复序列D3S1359基因座的多态性

应用PCR、聚丙烯酰胺凝胶垂直电泳及银染技术对STRD3S1359基因座进行多态性调查,首次获得中国汉族人群的基因频率分布数据。检出19个等位基因,59个基因型,基因型频率分布符合Hardy－Weinberg平衡。观察352次减数分裂未发现突变基因,DP值为0．9380,三联体PE值0．6311,二联体PE值0．4568,PIC值0．7824。证实D3S1359是一个高信息含量的STR标记系统。     

中国成都地区汉族群体5个STR基因座的遗传多态性

采用PCR扩增,聚丙烯酰胺凝胶电泳分析技术,调查中国成都汉族群体DIS1656、D851179、D9S302、D185535及D195253等5个STR基因座的等位基因频率分布。D1S1656检出11个等位基因,35种基因型;DSS1179检出9个等位基因,32种基因型;D95302检出12个等位基因,50种基因型;D185535检出7个等位基因,20种基因型;D195253检出8个等位基因,28种基因型。5个STR基因座基因型频率分布符合Hardy-weinberg平衡（P＞0．05）。个人识别机率（DP）为0．92～0．98。分析了二代3口之家的遗传模式,证明5个STR基因座均符合孟德尔遗传规律。5个STR基因座PCR扩增采用同一条件,方法简单、快速、灵敏、重复性好,可用于法科学亲子鉴定和个人识别。     

Allele and genotype frequencies for the STR locus D18S51 in a western German population

Genetic marker typing based on DNA amplification by the polymerase chain reaction (PCR) increasingly is being employed in forensic casework and for paternity testing. Allele frequencies were determined using PCR for 102 unrelated Germans (Rhine area) for the locus D18S51. Twelve alleles were observed, with frequencies ranging from 0.005 (allele 11) to 0.191 (allele 14). The observed heterozygosity was 0.867, and the power of discrimination was 0.968. There was no deviation from expectations under Hardy-Weinberg assumptions (P = 0.451).     

Evaluation of gastrointestinal cancer tissues as a source of genetic information for forensic investigations by using STRs

Malignant tissue samples may sometimes be the only source of biological material for forensic investigations, including identification of individuals or paternity testing. However, in use of such samples, uncertainties due to microsatellite instability (MSI) and loss of heterozygosity (LOH) often associated with neoplasias may be encountered. In this study, we have analysed the applicability of autosomal tetranucleotide short tandem repeat (STR) markers, which are routinely used in forensic analysis, to gain genetic information. MSI and LOH were analysed in 41 surgically removed gastrointestinal cancer specimens and the adjascent non-cancerous tissue marginals. The cancer specimens showed great variability in their genetic phenotypes due to MSI or LOH, with only 32% being microsatellite-stable. Of the 15 autosomal STR loci analysed, only TH01 had no MSI-type alteration in these samples. The loci most frequently affected by MSI were D8S1179, D21S11, D18S51 and D19S433 (MSI in 15-17% of cases). LOH-type alterations were observed at all of the loci, including the amelogenin locus used for sex determination. The highest LOH frequency was found at locus D18S51 (27%). The genetic alterations at the marker loci may indicate false homozygosity or heterozygosity, and false gender may result from erroneous deduction of DNA profiles. Therefore, typing of autosomal STRs from malignant tissues in forensic settings warrants careful interpretation of MSI and LOH results together with microscopic analysis of a tissue specimen. Results by two commercially available and widely used forensic DNA profiling kits used here were comparable.     

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.