Amplification of reproducible allele markers for amplified fragment length polymorphism analysis.

A procedure for amplification by PCR of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis is presented. We have prepared markers for the allelic products of the VNTR loci D1S80 (MCT118) and D17S30 (YNZ22) and for the hypervariable VNTR locus close to the 3' end of the apolipoprotein B gene (apoB) by re-amplifying a mixture of PCR products from individuals with known alleles. These allele markers allow precise and discrete determination of the VNTR alleles at these loci using the Amp-FLP technique that should prove suitable in forensic analyses, paternity testing and population studies.     

北京地区汉族群体D1S1612和D18S535基因座的遗传多态性

采用扩增片段长度多态性（Amp-FLP）分型技术,调查中国北京地区汉族群体D1S1612、D18S535 基因座的遗传多态性,获得等位基因频率分布。结果显示, D1S1612检出9个等位基因,25种基因型, D18S535检出9个等位基因,27种基因型。两个STR基因座的杂和度（H）分别为0.779、0.887;个人识别率（Dp）分别为0.901、0.927;非父排除率（PE）分别为0.564、0.770;多态信息容量（PIC）分别为0.723、0.796,卡方检验表明两个STR 基因座基因型频率分布符合Hardy-Weinberg平衡 (P>0.01 )。D1S1612和D18S535 基因座均属高杂合度、高识别能力的遗传标记,可用于法庭科学亲子鉴定和个人识别。 Abstract: To investigate the genetic polymorphism of D1S1612 and D18S535 in Han population of Beijing. Amp-FLP method was used. 9 alleles, 25 genotypes were observed for D1S1612 locus; and 9 alleles and 27 genotypes for D18S535 locus. All allele frequencies, heterozygosity (H), discrimination power (Dp), exclusion of paternity probability (PE) and polymorphism information content (PIC) were calculated. The allele distributions of the two loci were conformed to Hardy-Weinberg equilibrium (P>0.01). According to the results obtained in this study, it is suggested that both D1S1612 and D18S535 are useful genetic markers for individual identification and paternity testing in forensic science practice as well for genetic study.     

云南汉族人群D17S30位点扩增片段长度多态性A

应用PCR技术和小型聚丙烯酰胺凝胶电泳银染法, 对云南汉族人群D17S30位点扩增片段长度多态性进行了分析。在被检的105名无关个体中,共检出12个等位基因,41种基因型。等位基因频率范围在0.0048-0.2190之间,杂合度为83.81%,DP值为0.9647。观察的基因型分布符合Hardy-Weinber g定律。 Abstract:A study on amplified fragment length polymorphism(Amp-FLP)at locus D17S30 in Han nationality of Yunnan was carried out by using PCR followed by a high-resolution PAGE technique and silver staining.In a sample of 105 unrelated individuals,a total 12 different alleles and 41 genotypes were detected.The heterozygosity was 83.81% and the probability of discrimination(DP) was 0.9647.The distribution of observed genotypes obeyed the Hardy-Weinberg equilibrium.     

中国维吾尔族人群MSY1(DYF155S1)基因座多态性及其结构特点

应用荧光标记MVR-PCR、Amp-FLP与DNA序列分析技术等检测106例中国维吾尔族人群无关男性个体血纱样品,揭示了中国维吾尔族人群Y特异的小卫星MSY1 (DYF155S1)基因座5′和3′端多态性及其基因结构特点。DYF155S1基因座的多态性表现为3个方面：（1）长度多态性;（2）5′端多态性;（3）3′端多态性。106例无关个体共检出37个不同长度的片段,5′端检出68个类型,3′端检出23个类型。综合这3方面多态性,106例个体间没有相同,其基因多样性（h）超过0.9999。DNA序列分析发现该基因座5′端表现有7种模块结构,3′端有2种模块结构。DYF155S2片段缺失率约为4.7%。MVR-PCR、Amp-FLP与DNA序列分析技术结合起来可以更充分地揭示人群Y染色体特异的小卫星MSY1（DYF155S1）基因座多态性,并提出命名方式,从而为人类遗传学及法医学研究提供了有用的方法和基础资料。 Abstract:The study is to reveal the diversity and gene structure of 5′ and 3′ end of DYF155S1 locus in Y-chromosome minisatellite among Chinese Uygur population.Fluorescent MVR-PCR（minisatellite variant repeat by PCR）,Amp-FLP(Amplified fragment length polymorphism) and DNA sequencing methods were used repectively to detect 106 unrelated males among Chinese Uygur population.The polymorphisms of DYF155S1 locus could be revealed in three aspects:(1) polymorphic length:the sizes of amplified fragments ranged from 1405 to 2505bp.There are 37 types found among the 106 unrelated males.(2) polymorphism at 5′ end of DYF155S1 locus,68 types found among the 106 unrelated males.(3) polymorphism at 3′ end of DYF155S1 locus,23 types found among the 106 unrelated males.In combination of these three aspects of polymorphism,none of the 106 unrelated males tested had the same allele,and the gene diversity(h) was over 0.9999.Seven and two types of modular structure were founded in the 5′ and 3′ end of DYF155S1 locus,respectively,by DNA sequencing.The alleles at DYF155S2 locus showed yes/no dimorphism and the rate of deletion was 4.7%.The polymorphisms of DYF155S1 locus were fully revealed by using combination of MVR-PCR, Amp-FLP and DNA sequencing methods, and we suggested the nomenclature for alleles of MVR loci.These methods are useful tools and provide basic data for the study of human genetics and forensic medicine.     

Population Genetic Characteristics of the D1S80 locus in seven human populations

We have analyzed the allele frequency distribution at the highly polymorphic variable number of tandem repeat (VNTR) locus D1S80 (pMCT118) in seven ethnic populations (namely, New Guinea Highlanders of Papua New Guinea, Dogrib Indians of Canada, Pehuenche Indians of Chile, American and Western Samoans, Kacharis of Northeast India, and German Caucasians) using the polymerase chain reaction (PCR) technique. In the pooled sample of 443 unrelated individuals 20 segregating alleles were detected. A trimodal pattern of allelic distribution is present in the majority of populations and is indicative of the evolutionary antiquity of the polymorphism at this locus. In spite of the observed high degree of polymorphism (expected heterozygosity 56%–86%), with a single exception — the marginally significant P value (0.04) of the exact test in American Samoans — the genotype distributions in all populations conform to their respective Hardy-Weinberg expectations. Summary statistics indicate that, in general, the allele frequency distribution at this locus may be approximated by the infinite allele model. The data also demonstrate that alleles that are shared by all populations have the highest average frequency within populations. Furthermore, the kinship bioassay analysis demonstrates that the extensive variation observed at the D1S80 locus is at the interindividual within population level, which dwarfs any interpopulation allele frequency variation, consistent with the population dynamics of hypervariable polymorphisms. These characteristics of the D1S80 locus make it a very useful marker for population genetic research, genetic linkage studies, forensic identification of individuals, and for determination of biological relatedness of individuals.     

Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE.

Allelic data for the D1S80 locus was obtained by using the PCR and subsequent analysis with a high-resolution, horizontal PAGE technique and silver staining. Compared with RFLP analysis of VNTR loci by Southern blotting, the approach described in this paper offers certain advantages: (1) discrete allele resolution, (2) minimal measurement error, (3) correct genotyping of single-band VNTR patterns, (4) a nonisotopic assay, (5) a permanent record of the electrophoretic separation, and (6) reduced assay time. In a sample of 99 unrelated Caucasians, the D1S80 locus demonstrated a heterozygosity of 80.8% with 37 phenotypes and 16 alleles. The distribution of genotypes is in agreement with expected values according to the Hardy-Weinberg equilibrium. Furthermore, the observed number of alleles and the level of heterozygosity, obtained through the protocol described here, were congruent with each other in accordance with the expectation of a mutation-drift equilibrium model for a single, homogeneous, random-mating population. Therefore, the analysis of D1S80 and similar VNTR loci by amplified fragment length polymorphism (AMP-FLP) may prove useful as models for population genetic issues for VNTR loci analyzed by RFLP typing via Southern blotting.     

云南白族人群Y染色体DYF155S1基因座多态性研究

揭示云南白族136例无关男性个体Y染色体小卫星DYF155S1基因座多态性。用已建立的AmpFLP和MVRPCR方法分析DYF155S1基因座长度多态性、5′端多态性、3′端多态性。DYF155S1基因座具有高度多态性,基因多样性（h）达09996。研究表明将AmpFLP和荧光MVRPCR结合起来更能充分揭示DYF155S1基因座多态性,可为遗传学、人类学及法医学的研究提供有效的工具和基础资料。     

Genetic polymorphism study at four minisatellite loci (D1S80, D17S5, D19S20, and APOB) among five Indian population groups

The present study reports the genetic variation observed among five anthropologically distinct population groups of India, using four highly polymorphic minisatellite loci (D1S80, D17S5, D19S20, and APOB 3' VNTR) in order to examine the effect of geographical and linguistic affiliations on the genetic affinities among these groups. Random individuals from five ethnic groups were studied; the sample size ranged from 235 to 364. The population groups belong to two geographically separated regions of India, the state of Maharashtra (western India) and the state of Kerala (southern India). The two Maharashtrian groups (Konkanastha Brahmins and Marathas) speak "Marathi," an Indo-European language, whereas the three Kerala population groups (Nairs, Ezhavas, and Muslims) speak "Malayalam," an Indo-Dravidian language. Genomic DNA was extracted from peripheral blood samples and analyzed using amplified fragment length polymorphism (Amp-FLP) technique. All four loci displayed high heterozygosity with average heterozygosity in the range of 0.82 to 0.84. The Polymorphic Information Content and Power of Discrimination were > or = 0.75 and > or = 0.80, respectively. The coefficient of gene differentiation was found to be low (average G(ST) = 1.2%; range between 0.6% at D1S80 locus to 1.6% at APOB 3' VNTR locus) across the loci, indicating close affinity among the population groups. The neighbor-joining tree revealed two clear clusters, one for the two Maharashtrian population groups and the other for the three Kerala population groups. The results obtained are in conformity with the geographical and linguistic backgrounds of the studied populations.     

Genetic structure of the Sardinian population at the D1S80 VNTR locus and population diversity

We typed the Sardinian population at the D1S80 VNTR locus. Nineteen alleles were detected in a sample of 92 unrelated individuals, allele frequency distribution showing a modal pattern mostly in agreement with other Caucasoid populations. A high degree of heterozygosity (observed value=80.4%) was present. Goodness-of-fit tests demonstrated no departure from Hardy-Weinberg expectations. Data regarding heterozygosity, number of alleles and singletons appeared in accordance with the IAM mutation-drift equilibrium model and showed no evidence of hidden substructuring. Allele 34 exhibited in Sardinians the highest frequency never observed in Caucasians. Nonetheless, the comparison with other European populations did not disclose Sardinian genetic peculiarity. Indeed, measures of genetic divergence among Europeans demonstrated definitely smaller values at the D1S80 locus in comparison with those calculated over a high number of (pre-DNA) polymorphic loci. High mutation rate and selective neutrality typical of VNTRs could account for the observed moderate genetic divergence. Isolation and genetic drift, on the other hand, may have determined certain deviations in allele frequency distribution, as occurred to allele 34 in the Sardinian population.     

Allele frequency of VNTR locus D1S80 observed in Hb D-Los Angeles carrires

One of our previous studies presented the allele frequencies of D1S80 VNTR locus in province Denizli including the high frequencies of allele 24 and 18. In Denizli province of Turkey, the most common abnormal variant is Hb D-Los Angeles with a frequency of 57.8?% of the total abnormal Hbs. The aim of this study is to identify the allele frequencies of D1S80 VNTR locus in Hb D-Los Angeles carriers in Denizli province of Turkey. We studied unrelated 36 Hb D-Los Angeles carriers residing in Denizli province of Turkey. The size range of the D1S80 VNTR locus PCR products was determined first by agarose gel electrophoresis and then by a capillary electrophoresis system. For all subjects, DNA sequencing was performed. Allele frequency, theta (k) value, and observed and expected heterozygosity were calculated using Arlequin Software version 3.11. The most common alleles were the 24 (32?%), 18 (18.1?%) and 29 (16.7?%) alleles, and frequencies of these alleles were 0.329, 0.186 and 0.171 respectively. Other observed alleles percentages were 33, 2?%. We did not observe alleles 6, 15, 27 and 35, but we observed alleles 20 and 33. Results were in Hardy–Weinberg linkage disequilibrium. Observed heterozygosity was 0.889, and expected heterozygosity was 0.847. Theta (k) value was 4.91 (95?% confidence interval limits). According to our results, we concluded that Hb D-Los Angeles carriers have different allele frequencies in D1S80 VNTR and also have their own D1S80 VNTR locus divergence.     

Variable number of tandem repeat (VNTR) polymorphism at locus D17S5 (YNZ22) in four ethnically defined human populations

Summary We have analyzed the hypervariable locus D17S5 in four well-defined human populations (Kachari of Northeast India; Dogrib Indian of Canada; New Guinea Highlander of Papua New Guinea; and a relatively homogeneous Caucasian population of North German extraction) using both Southern blot analysis and the polymerase chain reaction (PCR) technique to; (1) compare the efficiency and limitation of Southern blotting versus PCR-based techniques in genotyping variable number of tandem repeat loci, and (2) provide allele frequency data at this locus in these four anthropologically defined populations. Preferential PCR amplification of smaller alleles associated with D17S5 was corrected by lowering the DNA template concentration to 200ng, and by reducing the extension time to 2 min. A perfect correspondence was observed between the results from Southern blot and PCR analysis in all but one sample. A very large allele, of approximately 24 to 25 repeat units, detected by Southern blotting, could not be amplified by PCR, resulting in an incorrect genotyping rate of less than 0.5%. Considering the grave consequences of mistyping in forensic and paternity testing, it is suggested that heterozygous controls consisting of large and small alleles should be employed in each PCR experiment, and PCR-generated homozygotes should be confirmed by Southern blotting. Significant variation in the number and frequency of alleles at this locus was observed in the four examined populations. A total of 15 different alleles were detected. The average heterozygosity varied from 54% in the Dogrib to 89% in the Kachari. No heterozygote deficiency was observed at this locus in any of the examined populations.     

Polymorphic distribution and forensic effectiveness study of eight miniSTR in Chinese Uyghur ethnic group

We obtained the allelic frequencies and forensic efficiency data for eight mini short tandem repeat loci including Penta E, D12S391, D6S1043, D2S1338, D19S433, CSF1PO, Penta D and D19S253 loci from a sample of 128 unrelated Uyghur individuals from China. The amplification products of the eight STR loci are <240 bp in size. A total of 94 alleles were observed and the corresponding allelic frequencies ranged from 0.0039 to 0.3438 in the present study. Observed genotype distributions for each locus do not show deviations from Hardy–Weinberg equilibrium expectations. The combined power of discrimination, combined power of exclusion and combined matching probability of the eight STR loci equaled to 0.999999999963373, 0.9997770 and 3.6627 × 10^?11, respectively. Because of the small fragment length of PCR products and the high degree of polymorphisms, the eight STR loci are highly beneficial for the forensic analysis of degraded DNA samples which are commonly observed in forensic cases. The STR data of the Uyghur group were compared with the previously published population STR data of other groups from different ethnic or areas, and significant differences were observed among these groups at some loci.     

江浙沪和哈尔滨地区汉族D17S30位点的多态性分布

Ｄ１７Ｓ３０是位于人类染色体１７ｄ１３．３、以７０ｂｐ为重复单位的,具有高度多态性的ＶＮＴＲ位点,本文采用作者报道的微量快速Ａｍｐ－ＦＬＰ分型技术,对１００名哈尔滨市北方汉族人和１１０名江浙沪南方汉族人作了Ｄ１７Ｓ３０位点分型,发现在北方汉族与江浙沪南方汉族之间等位基因频率分布并无显著差异,但可见Ａ１和Ａ７基因频率北高南低,Ａ４基因频率则为南高北代,提示存在南北汉族之间的分化。Ｄ１７Ｓ３０位点南北     

Short tandem repeat polymorphism of the D2S1242 locus in a Japanese population

Allele frequencies and sequence characteristics of the D2S1242 short tandem repeat (STR) locus were studied in a Japanese population sample. A total of 10 D2S1242 alleles and 34 genotypes were identified in 273 unrelated Japanese individuals. The five most common alleles detected had frequencies of over 10%. No deviations from Hardy-Weinberg equilibrium were found when the expected allele values were compared with the observed values. Sequence analysis of each allele showed a tetranucleotide polymorphism. Alleles 9 to 14 had different sequence structures than alleles 15 to 19. Allele 18 had a different sequence in the Japanese sample compared to an Austrian sample. The power of discrimination was 0.95. The present results demonstrate that the D2S1242 STR locus is a useful genetic marker in the Japanese population.     

Allele Frequency of VNTR Locus D1S80 Observed in Denizli Province of Turkey

The variable numbers of tandem repeats (VNTR) locus D1S80, located on chromosome 1 (1p35-36), has a repeat unit 16 bp in length, and different numbers of these repeat units have been observed for populations of different origins and ethnicity. We used a molecular identification method based on capillary electrophoresis separation to analyze D1S80 locus polymorphism among 74 subjects from Denizli province, Turkey, finding an amplified fragment length size of 379–635 bp. Allele repeat numbers were deduced from these sizes and sequence comparison. The most common alleles were repeat units 24 (34.3%) and 18 (22.4%), with frequencies of 0.414 and 0.207, respectively. Other alleles were 25 (7.86%), 28 (5.71%), 22 (4.25%), and 29 (2.86%). The allele with 23 repeat units was not observed. Results were in Hardy–Weinberg linkage disequilibrium. Observed heterozygosity was 0.614, and expected heterozygosity was 0.787. Theta(k) value was 4.86 (95% confidence interval limits). Capillary electrophoresis is a powerful approach for accurate identification of VNTR loci, especially for low base pair units like D1S80, for prenatal diagnosis, linkage analysis, forensic identification, paternity testing, anthropological research, and phylogenetic studies.     

Parentage analysis for three Romanian families by DNA-fingerprinting

In forensic medicine, DNA fingerprinting for human identification and paternity testing is becoming a necessary procedure. The genetic locus D1S80 (MCT118) with Hinf I polymorphism of its 5' flanking sequence, HUMTH01 and D21S11 have been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels or high-resolution SDS-PAGE. Analysis of these VNTR loci was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permitted good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another. The aim of this study includes three paternity testing cases; they are the first three human DNA-fingerprints performed in Romania.     

Comparative study of genetic variation at 15 STR loci in three isolated populations of the Bosnian mountain area

Fifteen autosomal STR loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX, and FGA) were studied in three geographically close but isolated populations from the Bosnian mountain area. The three villages are Bobovica, Dejcici, and Lukomir. DNA was obtained from 83 individuals, and the allele frequencies and genetic diversity among the three sample groups were compared. In addition, seven of the STR loci (CSF1PO, D13S317, D3S1358, D5S818, D7S820, FGA, TH01) were used in a comparative population analysis of the Bjelasnica-Treskavica region and the Adriatic islands of Brac, Hvar, and Korcula. Although the sample sizes are relatively small, the observed variation within any of the small isolated populations is high and comparable to less isolated groups. In addition, even though the populations are geographically isolated, the STR data are similar among the populations. The most significant frequency differences were observed at the TH01 locus. Although the specific allele distributions in any untyped population cannot be determined a priori, we find support for a high degree of diversity for the STR loci in most populations. In addition, the multiple locus profile is highly informative not only for various population studies but also for forensic studies, even when specific population data are not available.     

Assignment of the locus for PLO-SL, a frontal-lobe dementia with bone cysts, to 19q13.

PLO-SL (polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy) is a recessively inherited disorder characterized by systemic bone cysts and progressive presenile frontal-lobe dementia, resulting in death at <50 years of age. Since the 1960s, approximately 160 cases have been reported, mainly in Japan and Finland. The pathogenesis of the disease is unknown. In this article, we report the assignment of the locus for PLO-SL, by random genome screening using a modification of the haplotype-sharing method, in patients from a genetically isolated population. By screening five patient samples from 2 Finnish families, followed by linkage analysis of 12 Finnish families, 3 Swedish families, and 1 Norwegian family, we were able to assign the PLO-SL locus to a 9-cM interval between markers D19S191 and D19S420 on chromosome 19q13. The critical region was further restricted, to approximately 1.8 Mb, by linkage-disequilibrium analysis of the Finnish families. According to the haplotype analysis, one Swedish and one Norwegian PLO-SL family are not linked to the chromosome 19 locus, suggesting that PLO-SL is a heterogeneous disease. In this chromosomal region, one potential candidate gene for PLO-SL, the gene encoding amyloid precursor-like protein 1, was analyzed, but no mutations were detected in the coding region.     

The relevance of paternity analysis in Romanian population using the D1S80 locus

At present, DNA fingerprinting for human identification and paternity testing is a necessary and usual procedure. D1S80 is one of the best known polymorphic loci showing a VNTR, and exhibiting a high heterozygosity. This genetic locus, with a Tsp 509 I polymorphism of its 5' flanking sequence (1, 9), have been successfully amplified from human genomic DNA isolated from blood. The Tsp 509 I polymorphism was detected by restriction after PCR amplification. We tested the relevance of paternity analysis using the D1S80 locus considering the allele frequency distribution characteristic for our country. Paternal and maternal bands were compared with the children's DNA patterns. Our data include a comparison between D1S80 alleles amplified from mother, child and the supposed father for three tested families. This study was the first of this type made in Romania. We concluded a good power of discrimination and exclusion for this locus. It can be used successfully in the case of subtypes with low frequencies, and this is frequent for our population because of the high heterozygosity of D1S80 subtypes in Romanian population. We recommend the D1S80 use for exclusion paternity tests in Romanian population, as a very useful molecular tool, but we also recommend a complete set of molecular markers for confirmation paternity test in the same population.     

新疆4个民族STR基因座遗传多态性研究

对新疆维吾尔放族,锡伯族,乌孜别克族,柯尔克孜族4个民族的400份样本和40个家系进行STR基因扫描,基因分型和遗传结构分析。获得了4个民族STR遗传特征及遗传方式等的科学数据。结果为9个STR基因座上维吾尔族有66种STR等位基因,148种基因型;锡伯族有72种STR等位基因,163种基因型;乌孜别克族有65种TSR等位基因,168种基因型;柯尔克孜族有71种STR等位基因,191种基因型,用新疆4个民族的数据和汉族人群,美国高加索人群,美国黑人相比较发现,中国民族遗传特征数据之间差异不显著,而和国外民族相比差异显著,进一步证明中华民族是一个不可分割的大家庭。