Paternity testing and forensic DNA typing by multiplex STR analysis using ABI PRISM 310 Genetic Analyzer

Short tandem repeats (STRs) are widespread throughout the human genome and are a rich source of highly polymorphic markers which can be detected by PCR. To gain a better appreciation for how the polymorphism at a particular locus impacts the individual identity, the present study was undertaken to explore the use of 15 STR loci in forensic investigation and paternity testing. Multiplex STR typing was used to study the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) in addition to a gender identification marker, amelogenin, by capillary electrophoresis on 310 Genetic Analyzer. Samples from 85 trio and duo cases of disputed paternity were investigated. The data were analyzed to give information on paternity index, probability of paternity, frequency of number of exclusions and rate of mismatch at each STR locus. The method was also successfully applied to forensic personal identification in theft and murder cases. The results demonstrated that the STR typing is a reliable and robust tool for analyzing the forensic practice as well as for paternity testing. The advantages of using multiplex STR analysis over other conventional methods are discussed.     

湖南地区1013例亲子鉴定中的STR突变位点研究

对亲子鉴定常用的ABI公司Indentifiler荧光标记复合扩增试剂盒中的15个短串联重复序列及D14S306、D16S3391、D5S2500、D12S391、D13S796、D1S518位点的突变现象进行研究.在1013例认定亲子关系案例中,对发现有一个基因位点发生突变的案例增加8个常染色体STR（short tandem repeat）基因座检测,使其父权相对机会（RCP）大于99.999%以上,并对突变位点进行测序.在1013例认定亲子关系案例中,发现11例有一个基因位点发生突变,8次突变事件为父源性突变,突变位点包括vWA、FGA、D14S306、D13S317、D21S11、CSFIPO、D16S3391;其余3例突变来源不明,包括FGA、D13S796、D3S1358.以vWA和FGA的突变率最高,为0.15%,平均突变率为（0.09±0.370×10^-3）%.本鉴定所常用的21个基因座,突变率低,具有较高的推广价值.     

Nine polymorphic STR loci in the HLA region in the Shaanxi Han population of China

A large number of microsatellite genetic markers have been identified in the human leukocyte antigen (HLA) region. We investigated genetic polymorphism of the nine short tandem repeat (STR) loci (D6S276, MOGCA, D6S265, MIB, D6S273, G51152, TAP1CA, RING3CA, and D6S291) in the HLA region in the Shaanxi Han population. Using a fluorescence-labeled multiplex-PCR STR typing method, 6-13 alleles were detected in these nine STR loci in 150 unrelated Han Chinese from the region of Shaanxi, China. The distributions of the genotypes at these nine loci were in Hardy-Weinberg equilibrium. We conclude that these nine STR loci have a high level of genetic polymorphism; they would be useful for population genetic studies, pre-transplantation HLA typing, forensic and paternity testing, etc.     

Evaluation of 13 short tandem repeat loci for use in personal identification applications.

Personal identification by using DNA typing methodologies has been an issue in the popular and scientific press for several years. We present a PCR-based DNA-typing method using 13 unlinked short tandem repeat (STR) loci. Validation of the loci and methodology has been performed to meet standards set by the forensic community and the accrediting organization for parentage testing. Extensive statistical analysis has addressed the issues surrounding the presentation of "match" statistics. We have found STR loci to provide a rapid, sensitive, and reliable method of DNA typing for parentage testing, forensic identification, and medical diagnostics. Valid statistical analysis is generally simpler than similar analysis of RFLP-VNTR results and provides powerful statistical evidence of the low frequency of random multilocus genotype matching.     

Comparison of Biochemical Polymorphisms and Short Tandem Repeat (STR) DNA Markers for Paternity Testing in Rhesus Monkeys (Macaca mulatta)

Genetic markers are indispensable for molecular and statistical genetic research involving nonhuman primates. Genetic markers must be used to ascertain parentage and to confirm the accuracy of pedigrees based solely on housing or demographic records; otherwise, the results of pedigree, linkage, or quantitative genetic analyses may be unreliable. Until recently, most genetic markers used in nonhuman primates were plasma proteins or isozyme polymorphisms, which were required in large numbers, because levels of genetic variation revealed by these markers were rather low. We compared the newer, PCR-amplified short tandem repeat markers (STRs) with a panel of classical biochemical polymorphic markers, for paternity determination among captive-bred rhesus monkeys. The STR markers exhibited an average genetic diversity of 64% and an expected paternity exclusion probability of 0.443. Both of these were greater than the average 54.5% genetic diversity and 0.298 exclusion probability exhibited by the biochemical markers. The STRs were much more efficient than the biochemical markers for parentage determination, since they required only half the amount of genetic typing data to resolve an average paternity case. Thus, the results of applying these two classes of genetic markers in paternity tests were somewhat different than expected on the basis of theoretical exclusion probabilities. These differences were probably due to inbreeding and other genetic differences among breeding colonies. Because they are more informative and provide rapid and efficient genetic data, STRs are now the method of choice for parentage determination and pedigree corroboration among nonhuman primates.     

Validation of microsatellite markers for routine horse parentage testing

A parallel testing of 4803 routine Quarter Horse parentage cases, using 15 loci of blood group and protein polymorphisms (blood typing) and 11 loci of dinucleotide repeat microsatellites (DNA typing), validated DNA markers for horse pedigree verification. For the 26 loci, taken together, the theoretical effectiveness of detecting incorrect parentage was 99·999%, making it extremely unlikely that false parentage would fail to be recognized. The tests identified incorrect parentage assignment for 95 offspring (2% of cases). Despite fewer loci, DNA typing was as effective as blood typing and, in parentage exclusion cases, provided more systems to substantiate the genetic incompatibility. Five offspring presented potential genetic incompatibilities with their parents in only a single microsatellite system, but the parentage exclusions could not be confirmed with discordant results at additional loci. Two of these five incompatibilities could be explained as consequences of a null allele and three as fragment size increases or decreases (putative mutations). Provided that an exclusion assignment was based on at least two systems of genetic incompatibility, such rare genetic events did not lead to false exclusions. Notwithstanding the near 100% effectiveness estimations for either typing panel alone to identify incorrect parentage, this validation test showed an actual effectiveness of 97·3% for blood typing and 98·2% for DNA typing. The DNA-based test, however, may feasibly achieve higher efficacy than reported here by adding selected systems to the parentage test panel.     

广西环江县毛南族九个短串联重复序列基因座的遗传学分析

为了了解广西环江毛南族人群无关个体的九个短串联重复序列:vWA,D18S51,D5S818,FGA,D8S1179,D21S11,D7S820,D3S1358,D13S317基因座的遗传多态性分布情况;本文用枸橼酸钠抗凝法采集广西环江县毛南族200份无亲缘关系的健康个体的血样,Chelex-100方法提取DNA,应用AmpFlSTRIdentifilerTM荧光标记复合扩增技术对血样DNA的九个STR基因座进行扩增,用ABI 3100型遗传分析仪对扩增产物进行检测。结果显示九个STR位点的基因型分布均符合Hardy-Weinberg平衡定律,累积非父排除率达0.999996,累积个体识别能力达0.99999999996,多态信息总量为0.9999985。结论:广西环江县毛南族人群有自身的STR等位基因分布特征,所获数据可为法医学个体识别、亲子鉴定及群体的遗传学研究提供依据。     

Polymorphism profile of nine short tandem repeat Loci in the Han chinese

Nine short tandem repeat (STR) markers (D3S1358, VWA, FGA, THO1, TPOX, CSFIPO, D5S818, D13S317, and D7S820) and a sex-identification marker (Amel-ogenin locus) were amplified with multiplex PCR and were genotyped with a four-color fluorescence method in samples from 174 unrelated Han individuals in North China. The allele frequencies, genotype frequencies, heterozygosity, probability of discrimination powers, probability of paternity exclusion and Hardy-Weinberg equilibrium expectations were determined. The results demonstrated that the genotypes at all these STR loci in Han population conform to Hardy-Weinberg equilibrium expectations. The combined discrimination power (DP) was 1.05×10-10 within nine STR loci analyzed and the probability of paternity exclusion (EPP) was 0.9998. The results indicate that these nine STR loci and the Amelo-genin locus are useful markers for human identification, paternity and maternity testing and sex determination in forensic sciences.     

Microsatellite-based parentage control in cattle

As a new approach to parentage control we developed two multiplex coamplification polymerase chain reaction (PCR) systems containing a total of six different short tandem repeat (STR) loci; the microsatellite polymorphisms were visualized by automated fluorescence detection on the Applied Biosystems 373 DNA Sequencer with 672 Genescan Analysis software. Allele frequency data were determined from 238 animals. Thirty-five bovine parentage control cases not solvable by conventional blood typing could be solved.     

Selection and implementation of single nucleotide polymorphism markers for parentage analysis in crossbred cattle population

Crossbreeding is an essential way of improving herd performance. However, frequent parentage record errors appear, which results in the lower accuracy of genetic parameter estimation and genetic evaluation. This study aims to build a single nucleotide polymorphism (SNP) panel with sufficient power for parentage testing in the crossbred population of Simmental and Holstein cattle. The direct sequencing technique in PCR products of pooling DNA along with matrix-assisted laser desorption/ionization time-of-flight MS method for genotyping the individuals was applied. A panel comprising 50 highly informative SNPs for parentage analysis was developed in the crossbred population. The average minor allele frequency for SNPs was 0.43, and the cumulative probability of exclusion for single-parent and both-parent inference met 0.99797 and 0.999999, respectively. The maker-set for parentage verification was then used in a group of 81 trios with aid of the likelihood-based parentage-assignment program of Cervus software. Reconfirmation with on-farm records showed that this 50-SNP system could provide sufficient and reliable information for parentage testing with the parental errors for mother–offspring and sire–offspring being 8.6 and 18.5%, respectively. In conclusion, a set of low-cost and efficient SNPs for the paternity testing in the Simmental and Holstein crossbred population are provided.     

Characterization of <Emphasis Type="Italic">TPMT</Emphasis> minisatellite locus in five ethnic groups of India

A total of 206 random, healthy individuals belonging to five distinct ethnic groups (Ezhavas, Arayas, Nairs, Vishwakarmas and Muslims) were analyzed for 18 bp VNTR repeat polymorphism present in the 5í flanking region of the Thiopurine Methyl Transferase gene (TPMT). In the present study, the population data of TPMT minisatellite was compared with the population data of other loci and the utility of minisatellite was evaluated in population studies. Human tandem repeat alleles of the TPMT minisatellite locus were characterized for the length polymorphism. The expected and observed heterozygosity did not show any significant difference. All five populations were in Hardy-Weinberg equilibrium. High polymorphism information iontent (PIC) (≥0.658) and power of discrimination (PD) (ranging from 0.775–0.860) value of this VNTR showed that this marker is informative. The combined power of discrimination of TPMT minisatellite along with other two loci studied earlier in our lab was 0.9964. The paternity exclusion power (PE) of TPMT VNTR ranged from 0.203 to 0.533 and the combined power of paternity exclusion (with two other loci D8S315 and D2S1328) was ≥0.8285. All these parameters (heterozygosity, PD, PIC, PE) of TPMT minisatellite locus showed that this marker is informative and can be used for DNA typing and population studies besides being used in clinical investigation in checking thiopurine drug sensitivity of individuals. The text was submitted by the autor in English.     

Genetic maternity and paternity in a local population of armadillos assessed by microsatellite DNA markers and field data

Genetic data from polymorphic microsatellite loci were employed to estimate paternity and maternity in a local population of nine-banded armadillos (Dasypus novemcinctus) in northern Florida. The parentage assessments took advantage of maximum likelihood procedures developed expressly for situations when individuals of neither gender can be excluded a priori as candidate parents. The molecular data for 290 individuals, interpreted alone and in conjunction with detailed biological and spatial information for the population, demonstrate high exclusion probabilities and reasonably strong likelihoods of genetic parentage assignment in many cases; low mean probabilities of successful reproductive contribution to the local population by individual armadillo adults in a given year; and statistically significant microspatial associations of parents and their offspring. Results suggest that molecular assays of highly polymorphic genetic systems can add considerable power to assessments of biological parentage in natural populations even when neither parent is otherwise known.     

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.     

A large population genetic study of 15 autosomal short tandem repeat loci for establishment of Korean DNA Profile Database

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10⁻¹⁷. This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications.     

Mexican mestizo population sub-structure: effects on genetic and forensic statistical parameters

Since Mexican mestizos are an admixed population, it is necessary to determine the effects that the substructure of the population has on genetic and forensic parameters. With this aim, a study was performed with 15 STR loci (CODIS plus D2S1338 and D19S433) on 1,640 unrelated Mexican mestizos. We determine allele and genotypic frequencies observing departure from Hardy–Weinberg expectation (12 out of 15 loci, with an excess of homozygotes, Fis?>?0), as well as pairs of loci in an apparent linkage disequilibrium (13 of 92 loci). We conducted a test for genetic population stratification, the results show that the Mexican mestizo population is substructured into three subgroups, which are in HW and linkage equilibrium. The combination of the 15 loci in the whole population has high forensic efficiency with the capacity to genetically discriminate one individual in one quintillion (1/10¹⁸). Our data potentially validates the use of these 15 STR loci to establish forensic identity and parentage testing for legal purposes, and offers a powerful tool for genetic variation analysis. However, given that the population is stratified, we highly recommend applying a correction with the inbreeding coefficient in calculations of paternity and forensic studies to avoid erroneous assumptions.     

中国南方汉族人群6个Y-STR基因座 遗传多态性及法医学应用

为研究中国南方汉族人群DYS393等6个Y-STR基因座的遗传多态性并用于法医学鉴定,通过采用PCR复合扩增和基因测序仪荧光检测方法,检查204个无关男性个体,调查南方汉族的6个Y-STR基因座的单倍型频率,并对93对真父子和38对非父子的亲子鉴定样本进行检测。结果DYS393基因座检出5个等位基因,DYS19基因座检出6个等位基因,DYS389Ⅱ基因座检出8个等位基因,DYS390基因座检出6个等位基因,DYS391基因座检出4个等位基因,DYS385 基因座检出44个等位基因,共检出176种单倍型。93对真父子中,观察到2例分别有1个基因座突变。检测38对非父子,有1个或2个Y-STR基因座排除的案例各有1例（2.6%）;有3 个和3个以上的Y-STR基因座可以排除父子关系的案例为35例（92.1%）;6个Y-STR基因座不能排除父子关系的为1例。结果表明6个Y-STR基因座具有丰富的遗传多态性,可用于法医学个体识别和亲子鉴定。Abstract: To study the genetic polymorphisms of six Y-chromosome specific STR loci in the southern Chinese Han population and apply it in forensic science, six Y-STR loci were amplified by multiple PCR and the PCR products were detected by using ABI PrismTM 377 Sequencer. The haplotype frequencies at 6 Y-STR loci were determined in a total of 204 unrelated males from southern Han population of China. Ninety-three father/son pairs with demonstrated paternity and thirty-eight non-paternity father/son pairs were detected by using our Y-STR system. As a result, the number of alleles for DYS393、DYS19、DYS389Ⅱ、DYS390、DYS391and DYS385 were 5, 6, 8, 6, 4 and 44 , respectively. A total of 176 haplotypes at 6 Y-STR loci were found. Two father/son pairs with single Y-STR mutation were observed in the 93 father/son pairs with demonstrated paternity. Among the 38 non-paternity father/son pairs, one case with one Y-STR exclusion of paternity, one case with two Y-STR exclusions and 35 cases with 3 or more Y-STR exclusions were observed. Non-exclusion of paternity at 6 Y-STR loci was found only in one case. This result indicated that the six Y-STR loci were highly polymorphic and are suitable for personal identification and paternity testing.     

Theoretical exclusion percentages obtainable by blood grouping of pigs

On the basis of the frequency of blood group alleles in Danish Landrace pigs, the theoretical probability of excluding incorrectly alleged paternity and parentage by means of the various blood group systems are calculated. The probability of excluding incorrect paternity of 1 and 4 pigs, will be 85 and 97%, respectively. In 96% of the cases, the parentage of an accidentally interchanged pig can be excluded.
A study of the progeny groups actually excluded during the period 1960-1968, showed good agreement between the observed and theoretically expected frequencies of exclusions in the various blood group systems, except for the G-system.     

Genetic diversity and assessment of 23 microsatellite markers for parentage testing of Santa Inês hair sheep in Brazil

Santa Inês is the most common hair sheep breed in Brazil and probably has the highest genetic diversity among sheep breeds in this country. Successful breeding programs for Brazilian sheep breeds are not common for various reasons, including a lack of control of parentage in the flocks. We developed an allele frequency database for 23 STR loci for the Santa Inês breed based on 285 animals sampled from five populations distributed across the central-western and north-eastern regions of Brazil. The marker set included seven microsatellites used in the 2011 International Society for Animal Genetics sheep genotyping comparison tests and all eight microsatellites currently approved by the Brazilian Agricultural Ministry laboratory accreditation guidelines for sheep identification. The microsatellites had an average of 10 alleles and a mean expected heterozygosity of 0.745. Combined paternity exclusion probabilities when no parent or one parent was known were >99.99%. A small proportion (5.8%) of the existing genetic variation was found to be among the Santa Inês populations, possibly derived from genetic drift and selection. We found that the marker panel proposed by the Agricultural Ministry, although generally useful, should be enhanced by including more markers for improved exclusionary power in parentage testing. This database provides a useful tool for parentage testing of this major Brazilian breed, contributing to improved management and breeding of existing herds.     

用多重PCR检测上海地区汉族人群9个STR基因座的多态性

利用多重PCR和四色荧光（5－FAM,JOE,NED和ROX）自动化检测技术调查上海地区汉族人群D3S1358、vWA、FGA、D8S1179、D21S11、D18S51、D5S818、D13S317、D7S820等9个STR基因座多态性分布并计算 该9个基因座的的基因频率（Pi)、个体鉴别力（DP）、无偏倚期望杂合性（H）、多态性信息含量（PIC）和非父排除概率（PE）。结果显示：9个STR基因座的基因型分布符合Hardy-Weinberg平衡,9个STR基因座中FGA基因座的DP值最高为0.9584,D8S1179的H值最高为0.9403,D18S51的PIC值最高为0.8560,D18S51的PE值最高为0.7391,9个STR基因座累积个体鉴别力（CDP)为0.9999996,累积非父排除能力（CPE）为0.99991。9个STR基因座适合作为中国人群的遗传标志,用于人类学、遗传疾病基因连锁分析、法医学亲子鉴定和个体识别等研究领域。     

First searchable database for DNA profiles of human cell lines: sequential use of fingerprint techniques for authentication.

The authenticity and freedom from cross-contaminants of a cell line are important prerequisites for any research, development or production programs involving cell lines. Mini- and microsatellites in the human genome harboring variable-numbers of tandem repeat (VNTR) DNA markers allow individualization at the DNA level and are of practical value for genetic linkage mapping, forensic legal medicine, paternity testing, monitoring of bone marrow transplants, and individualization of established cell lines. We have validated fingerprint techniques of different single- and multiple-locus VNTRs enabling the establishment of a searchable database of DNA profiles. As a result, multiplexed polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of four prominent and highly polymorphic minisatellite VNTR loci was proven as the best tool for screening the uniqueness of DNA profiles in a fingerprint database. In order to avoid false positivity, identical or similar DNA profiles based on AmpFLP VNTR were tested further using a multi-locus fingerprint system. Our data demonstrate that misidentification remains a chronic problem among human continuous cell lines (detailed information at URL http://www.dsmz.de). The combination of rapidly generated DNA profiles based on single-locus VNTR loci, their authentication by screening the fingerprint database, and confirmation of duplicate banding patterns using multilocus fingerprints constitute a highly reliable and robust method, which enables high fidelity and quality of maintenance independent from the quantity of individual cell lines.