PCR产物纯化增强STR分型强度的研究

目的研究PCR产物纯化对微量DNA的STR分型的影响。方法使用25pg/μL的9947标准基因组DNA为模板,标准程序扩增和STR分型检测。设立对照组A和实验组B、C、D。实验组分别使用分子筛(Qiagen DyeEX2.0spin kit)、超滤膜(Amicon Ultra-0.5,100KD)、亲和层析(Qiagen MinElute Column)3种DNA纯化方法,对照组不做任何处理。结果与对照组相比,3种方法均可显著提高STR分型强度(P峰高<0.001),平均峰高约为对照组的4倍,并且3种方法对提高STR分型强度无显著差异(P峰高=0.249)。结论 PCR产物纯化能显著提高微量DNA的STR分型强度,可用于骨骼、脱落细胞等微量DNA检材的检验。     

Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples

To assist the interpretation of STR DNA typing results from forensic casework samples containing mixtures, the range of heterozygous allele peak height and peak area ratios (HR) and stutter percentages (stutter %) for the loci comprised in the AmpFlSTR Profiler Plus (PP) kit were assessed on 468 database and 275 casework single source samples. Stutter % medians were similar for database and casework samples, ranging from 2% to 7%. The upper limit of the stutter value range was 16%, calculated as median +3 SD, although lower locus-specific values could be used. HR medians were 93 +/- 6.5% for database samples, 88 +/- 12% for casework samples. For casework samples, the maximum signal imbalance noted was 52%, calculated as median -3 SD. No significant difference was observed between peak height and peak area calculated values. This study shows the importance of selecting the proper reference database for the establishment of HR threshold values.     

Identification and separation of DNA mixtures using peak area information

We introduce a new methodology, based upon probabilistic expert systems, for analysing forensic identification problems involving DNA mixture traces using quantitative peak area information. Peak area is modelled with conditional Gaussian distributions. The expert system can be used for ascertaining whether individuals, whose profiles have been measured, have contributed to the mixture. It can also be used to predict DNA profiles of unknown contributors by separating the mixture into its individual components. The potential of our probabilistic methodology is illustrated on case data examples and compared with alternative approaches. The advantages are that identification and separation issues can be handled in a unified way within a single probabilistic model and the uncertainty associated with the analysis is quantified. Further work, required to bring the methodology to a point where it could be applied to the routine analysis of casework, is discussed.     

AMPFlSTR Identifiler STR allele frequencies in Tanzania, Africa

POPULATION:  Identifiler—Employees and students of Muhimibili University College of Health Sciences in Dar es Salaam representing 19 widely distributed administrative districts and 42 tribes within the country.     

Interpreting DNA mixtures in structured populations.

DNA profiles from multiple-contributor samples are interpreted by comparing the probabilities of the profiles under alternative propositions. The propositions may specify some known contributors to the sample and may also specify a number of unknown contributors. The probability of the alleles carried by the set of people, known or unknown, depends on the allelic frequencies and also upon any relationships among the people. Membership of the same subpopulation implies a relationship from a shared evolutionary history, and this effect has been incorporated into the probabilities. This acknowledgment of the effects of population structure requires account to be taken of all people in a subpopulation who are typed, whether or not they contributed to the sample.     

Statistical analysis of DNA mixtures using peak area information and allelic drop out

A case of a woman killed in Perugia is reported. The woman was beaten to death and the body showed evidence of bites, kicks and punches. The request of the Court was to verify the presence of bites and if they belonged to humans. Morphological examination and genetic analysis with human Y-specific markers were performed in order to verify the origin of the bites. The DNA profile from the surrounded area of the traces was compared with the profile of the victim's husband (the suspect).The results showed a match between the profile of the suspect and that of the traces for all loci examined. Due to the fact that also other relatives of the husband's male lineage lived in the same house, it was not possible to identify the man who really contributed to the traces. Therefore, the analysis was implemented with autosomal STR markers, which showed a mixed genetic profile. In order to verify the number and the identity of the contributors, statistical analysis based upon peak area information was performed with Probabilistic Expert Systems.     

PENDULUM--a guideline-based approach to the interpretation of STR mixtures

Several years ago, a theory to interpret mixed DNA profiles was proposed that included a consideration of peak area using the method of least squares. This method of mixture interpretation has not been widely adopted because of the complexity of the associated calculations. Most reporting officers (RO) employ an experience and judgement based approach to the interpretation of mixed DNA profiles. Here we present an approach that has formalised the thinking behind this experience and judgement. This has been written into a computer program package called PENDULUM. The program uses a least squares method to estimate the pre-amplification mixture proportion for two potential contributors. It then calculates the heterozygous balance for all of the potential sets of genotypes. A list of "possible" genotypes is generated using a set of heuristic rules. External to the programme the candidate genotypes may then be used to formulate likelihood ratios (LR) that are based on alternative casework propositions. The system does not represent a black box approach; rather it has been integrated into the method currently used by the reporting officers at the Forensic Science Service (FSS). The time saved in automating routine calculations associated with mixtures analysis is significant. In addition, the computer program assists in unifying reporting processes, thereby improving the consistency of reporting.     

亲子鉴定中STR基因座的基因突变分析

目的探讨Identifiler^TM荧光标记复合扩增试剂盒15个STR基因座在亲子鉴定中的基因突变特点。方法应用Identifiler^TM荧光标记复合扩增试剂盒检测676例亲子鉴定案，对其中1～2个突变基因座加做HLA等位基因检测或Y—STR基因座检测。结果在认定亲子关系的676例中，观察1304次减数分裂，Identifiler^TM荧光标记复合扩增试剂盒中的15个基因座确定19例突变，其中D18S51基因座4例，D2S1338基因座3例，D8S1179、D16S539、vWA、D7S820、D13S317基因座各2例，D5S818和TH01基因座各1例，D21S11、FGA、D3S1358、D19S433、TPOX、CSF1P0基因座未见突变；一步突变的17例，二步突变的为1例，四步突变的1例；1个基因座发生突变的18例，2个基因座同时发生基因突变的为1例；突变来自父亲与来自母亲的比例为13：2，4例来源不能确定。结论用Identifiler^TM荧光标记复合扩增试剂盒检测到1—2个基因座发生突变，须增加对其它遗传标记的检测。     

Empirical analysis of the STR profiles resulting from conceptual mixtures

Samples containing DNA from two or more individuals can be difficult to interpret. Even ascertaining the number of contributors can be challenging and associated uncertainties can have dramatic effects on the interpretation of testing results. Using an FBI genotypes dataset, containing complete genotype information from the 13 Combined DNA Index System (CODIS) loci for 959 individuals, all possible mixtures of three individuals were exhaustively and empirically computed. Allele sharing between pairs of individuals in the original dataset, a randomized dataset and datasets of generated cousins and siblings was evaluated as were the number of loci that were necessary to reliably deduce the number of contributors present in simulated mixtures of four or less contributors. The relatively small number of alleles detectable at most CODIS loci and the fact that some alleles are likely to be shared between individuals within a population can make the maximum number of different alleles observed at any tested loci an unreliable indicator of the maximum number of contributors to a mixed DNA sample. This analysis does not use other data available from the electropherograms (such as peak height or peak area) to estimate the number of contributors to each mixture. As a result, the study represents a worst case analysis of mixture characterization. Within this dataset, approximately 3% of three-person mixtures would be mischaracterized as two-person mixtures and more than 70% of four-person mixtures would be mischaracterized as two- or three-person mixtures using only the maximum number of alleles observed at any tested locus.     

Accurate STR allele designations at the FGA and vWA loci despite primer site polymorphisms

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.     

二组分混合DNA样品STR图谱解释

对混合样品STR图谱的结果进行解释。实验模拟二组分DNA混合样品 ,复合扩增荧光检测 10个基因座 ,比较混合样品谱带 ,计算等位基因峰面积比。结果发现 :二组分DNA混合样品的等位基因数增加 ,样品的混合比例不同就出现峰面积的不平衡。在等位基因峰面积比值与样品组分混合比例接近时 ,可由峰面积比值推断混合样品的混合比例。在混合比例为 1∶2 0时 ,基本上检测不到来自少量混合成分的等位基因 ,表现为单一组分图谱 ;在混合比例为 1∶10时 ,含量低的组分的等位基因峰面积接近与主要组分的“Stutter”峰面积 ,与来自主要组分的等位基因峰面积差异很明显。能检出混合样品中少量成分等位基因的最高混合比例为 1∶10     

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.     

联合应用多个DNA位点进行尸源鉴定

目的　研究联合应用多个DNA位点在尸源鉴定方面的应用价值。方法　应用聚合酶链反应(PCR)、聚丙烯酰胺凝胶电泳分离及银染显带的方法通过对无名尸体的有关检材与可疑双亲或子女进行亲权鉴定。结果　在 80例刑事案件尸源鉴定中 ,38例无名尸体采用较新鲜肌肉 ,通过扩增VNTR、STR多个位点得以判明尸源 ;2 9例采用腐败肌肉、 6例采用骨骼和 2例采用牙齿 ,通过扩增多个STR位点判明了尸源。仅有 1例采用腐败肌肉、 2例采用骨骼未能确定尸源。结论　运用多个位点进行亲权方法可以准确地判明尸源鉴定 ,特别对高度腐败尸体、尸块不全等情况下的尸源鉴定具有很高的实用价值     

联合应用多个DNA位点进行尸源鉴定

目的 研究联合应用多个ＤＮＡ位点在尸源鉴定方面的应用价值。方法 应用聚合酶链反应（ＰＣＲ）、聚丙烯酰胺凝胶电泳分离及银染显带的方法通过对无名尸休的有关检材与可疑双亲或子女进行亲权鉴定。结果 在８０例刑事案件尸源鉴定中,３８例无名尸体采用较新鲜肌肉,通过扩增ＶＮＴＲ、ＳＴＲ多个位点得以判明尸源;２９例采用腐败肌肉、６例采用骨骼和２例采用牙齿,通过扩增多个ＳＴＲ位点判明了尸源。仅有１例采用腐败肌肉、２     

STR analysis of degraded DNA using a miniplex

Analysis of short tandem repeat (STR) markers currently represents the most useful instrument in the field of forensic genetics. The problem with forensic material is the degradation of the sample material. In recent years, several papers have demonstrated that short amplicon STR (miniSTR) represents one of the most useful tools for analyzing degraded DNA samples.In the present study, we attempted to develop a short amplicon STR multiplex system (autosomal and y-chromosomal) for analyzing degraded DNA using some newly designed primer sets for a multiplex polymerase chain reaction (PCR) systems for typing.An assay of degraded DNA samples using the designed multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. Comparing the multiplex with commercial kits, first results show a well success rate.     

Typing Y chromosome STR haplotypes using redesigned primers

A panel of Y-specific STR loci, including DYS19, DYS389, DYS390, DYS391, DYS392, and DYS393 was analyzed using horizontal nondenaturing polyacrylamide gel electrophoresis with a discontinuous buffer system (horizontal disk-PAGE). In order to obtain correct results for the larger DYS389 and DYS392 alleles, it was necessary to design new primers that bind closer to the repeat region and lead to a significant reduction of the amplified fragment size. Using the modified primer sets the horizontal disk-PAGE results were consistent with a nondenaturing approach using fluorescent primers and a 377 automated sequencer. The modified procedure also amplifies the second repeat stretch at the duplicated DYS389 locus as a single fragment, which results in an immediate allele identification. The results indicate that horizontal disk-PAGE with silverstaining is a simple approach to type the recommended Y-specific STR markers.     

Inferring recent human phylogenies using forensic STR technology

STR loci are characterized by extremely high mutation rates and thus, high levels of length polymorphism both within and among populations. In addition, much of the observed variation is believed to be nearly selectively neutral. Because of these features, STRs are ideal markers for genetic mapping, intra-species phylogenetic reconstructions and forensic analysis. In the present study, we investigate the application of five STR loci (CS1PO, TH01, TPOX, FGA and vWA) routinely used in forensic analysis for delineating the phylogenetic relationships of 10 human populations representing the three major racial groups (African-Caribbean, Croatian from the island of Hvar, East Asian, Han Chinese, Italian, Japanese, Portuguese, UK Caucasian, US Caucasian and Zimbabwe). The resulting tree topology exhibited strong geographic and racial partitioning consistent with that obtained with mtDNA haplotypes, Y-chromosome markers, SNPs, PAIs (polymorphic Alu insertions) as well as classic genetic polymorphisms. These findings suggest that forensic STR loci may be particularly powerful tools and provide the necessary fine resolution for the reconstruction of recent human evolutionary history.