Significance of mitochondrial DNA for forensic stain analysis, identification and forensic lineage determination

DNA testing using conventional STR systems may produce insufficient results, if the genomic DNA in the specimen is either highly degraded or the available quantity is very small (e.g. skin particles, hair shafts or ancient bones). In some of these cases the examination of mitochondrial DNA, which is present in considerably larger copy numbers in the cytoplasm, is more successful than that of nuclear DNA. Identification of unknown corpses by conventional DNA typing sometimes remains doubtful, if only samples from presumably distant relatives or putative brothers or sisters are available for comparison. Since mitochondrial DNA is generally transmitted in maternal lineages, its sequence pattern can be directly compared with those of other individuals and, in case of the same maternal lineage, corresponding sequence chromatograms are to be expected. In connection with nuclear DNA typing methods certain sequence motives may furnish clues to ethnic groups. The report presents three cases illustrating the application possibilities of mtDNA typing in forensic practice.     

纳米科技与法医DNA检验

综述了纳米科技进步将对法医DNA检验产生的深远影响,对从DNA提取到基因芯片研究等多个未来研究的新方向进行了探讨。     

Sample collection strategies when building mitochondrial DNA forensic databases

For establishing databases that capture the existing diversity in populations, the sample collection strategy is a determining factor and caution must be taken when choosing the suitable approach. Many researchers choose to restrict the sampling to individuals with inheritance for three generations in a specific geographic location. However, the appropriate database in a forensic context is the one representing the current population. We analyzed mtDNA composition across generations in populations from Colombia, Ecuador, and Paraguay. An overall genetic homogeneity was detected, with statistically significant differences on macrohaplogroup frequencies for few department/regions.     

Recovery of DNA for forensic analysis from lip cosmetics.

To obtain a reference DNA profile from a missing person, we analyzed a variety of personal effects, including two lip cosmetics, both of which gave full DNA profiles. Further investigations were undertaken to explore this previously unreported source of DNA. We have tested a range of brands and types of lip cosmetics. Our studies have revealed that lip cosmetics are an excellent source of DNA, with almost 80% of samples giving a result. However, artifacts are frequently observed in the DNA profiles when Chelex is used for the DNA extraction and additional DNA purification procedures are required to ensure that an accurate DNA profile is obtained.     

Use of subpopulation data in Australian forensic DNA casework

DNA profiling evidence presented in court should be accompanied by a reliable estimate of its evidential weight. In calculating such statistics, allele frequencies from commonly employed autosomal microsatellite loci are required. These allele frequencies should be collected at a level that appropriately represents the genetic diversity that exists in the population. Typically this occurs at broadly defined bio-geographic categories, such as Caucasian or Asian. Datasets are commonly administered at the jurisdictional level. This paper focuses on Australian jurisdictions and assesses whether this current practice is appropriate for Aboriginal Australian and Caucasian populations alike. In keeping with other studies we observe negligible differences between Caucasian populations within Australia when segregated geographically. However segregation of Aboriginal Australian population data along contemporary State and Territory lines appears to mask the diversity that exists within this subpopulation. For this reason datasets collated along more traditional lines may be more appropriate, particularly to distinguish the most genetically differentiated populations residing in the north of the continent.     

A PCR multiplex and database for forensic DNA identification of dogs

Animal-derived trace evidence is a common finding at crime scenes and may provide an important link between victim(s) and suspect(s). A database of 558 dogs of pure and mixed breeds is described and analyzed with two PCR multiplexes of 17 microsatellites. Summary statistics (number of alleles, expected and observed heterozygosity and power of exclusion) are compared between breeds. Marked population substructure in dog breeds indicates significant inbreeding, and the use of a conservative theta value is recommended in likelihood calculations for determining the significance of a DNA match. Evidence is presented that the informativeness of the canine microsatellites, despite inbreeding, is comparable to the human CODIS loci. Two cases utilizing canine DNA typing, State of Washington v. Kenneth Leuluaialii and George Tuilefano and Crown v. Daniel McGowan, illustrate the potential of canine microsatellite markers for forensic investigations.     

A DNA microarray system for forensic SNP analysis

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell. Due to the increasing number of complete mtDNA genome sequences available, the limited discrimination power of an mtDNA analysis, may be increased by analysis of coding region polymorphisms in addition to the non-coding variation. Since sequence analysis of the coding region would require more material than generally present in forensic samples, an alternative SNP analysis approach is required. We have developed a one-colour microarray-based SNP detection system for limited forensic materials. The method is based on minisequencing in solution prior to hybridisation to universal tag-arrays. In a first outline of a forensic chip, a combination of 12 nuclear and 21 mitochondrial SNP markers are analysed simultaneously. The mitochondrial markers on the chip are polymorphisms within the hypervariable region as well as in the coding region. Even though the number of markers in the current system is limited, it can easily be extended to yield a greater power of discrimination. When fully developed, microarray analysis provides a promising system for efficient sensitive SNP analysis of forensic samples in the future.     

DNA甲基化在法医DNA分析中的应用

表观遗传学在生命的发生、发展过程中起着十分重要的作用。DNA甲基化作为表观遗传的一个重要方面,不仅参与多种基因的表达调控,与机体的发育、肿瘤发生等密切相关,而且具有可遗传性、相对稳定性、亲缘特异性、基因组中含量丰富等特点,已证实适用于法医DNA分析。本文对近年来DNA甲基化在印迹基因、同卵双生子鉴定、年龄、性别推断方面的研究与应用进行回顾与综述,以期为在法医学及相关领域中应用提供参考。     

法医DNA分析的其他应用

综述了法医DNA的多种用途,如人死后根据体内DNA变化情况推断死后时间间隔,利用线粒体DNA4977bp的缺失以及端粒的长度推断年龄;利用昆虫mtDNA的细胞色素氧化酶亚基I进行昆虫种属鉴定,以帮助确定人的腐尸死亡时间;利用一些食血昆虫体内的人DNA帮助了解与案件的关系;利用DNA标记进行动物如犬、牛、马及猪等个体识别与亲子鉴定,解决因动物引起的纠纷,或者利用DNA分析嫌疑人周围的动物毛发、植物及土壤微生物等,确定嫌疑人与案件的关系。     

Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis

A modified alkaline lysis protocol for extracting DNA from forensically relevant specimens is evaluated and compared with the chelex 100 method. For whole blood, bloodstains and sperm stains, both methods yielded comparable results after amplification for a pentameric STR locus (HumCD4). The main advantages of the new method are: only approximately ten minutes and two pipetting steps are necessary and the expenses for the extraction are extremely low as only NaOH, TrisHCl buffer and a single microcentrifuge tube are required. Alkaline lysis also proved to yield DNA suitable for typing longer STRs by using dye-labeled primers and capillary electrophoresis. These advantages should render this protocol especially interesting for high-throughput laboratories in combination with multiplex PCR and fluorescent dye technology, although the storability of the extracts proved to be problematic.     

Choice of population database for forensic DNA profile analysis

When evaluating the weight of evidence (WoE) for an individual to be a contributor to a DNA sample, an allele frequency database is required. The allele frequencies are needed to inform about genotype probabilities for unknown contributors of DNA to the sample. Typically databases are available from several populations, and a common practice is to evaluate the WoE using each available database for each unknown contributor. Often the most conservative WoE (most favourable to the defence) is the one reported to the court. However the number of human populations that could be considered is essentially unlimited and the number of contributors to a sample can be large, making it impractical to perform every possible WoE calculation, particularly for complex crime scene profiles. We propose instead the use of only the database that best matches the ancestry of the queried contributor, together with a substantial F_ST adjustment. To investigate the degree of conservativeness of this approach, we performed extensive simulations of one- and two-contributor crime scene profiles, in the latter case with, and without, the profile of the second contributor available for the analysis. The genotypes were simulated using five population databases, which were also available for the analysis, and evaluations of WoE using our heuristic rule were compared with several alternative calculations using different databases. Using F_ST = 0.03, we found that our heuristic gave WoE more favourable to the defence than alternative calculations in well over 99% of the comparisons we considered; on average the difference in WoE was just under 0.2 bans (orders of magnitude) per locus. The degree of conservativeness of the heuristic rule can be adjusted through the F_ST value. We propose the use of this heuristic for DNA profile WoE calculations, due to its ease of implementation, and efficient use of the evidence while allowing a flexible degree of conservativeness.     

FaSTR DNA: A new expert system for forensic DNA analysis

The automation of DNA profile analysis of reference and crime samples continues to gain pace driven in part by a realisation by the criminal justice system of the positive impact DNA technology can have in aiding in the solution of crime and the apprehension of suspects. Expert systems to automate the profile analysis component of the process are beginning to be developed. In this paper, we report the validation of a new expert system FaSTR DNA, an expert system suitable for the analysis of DNA profiles from single source reference samples and from crime samples. We compare the performance of FaSTR DNA with that of other equivalent systems, GeneMapper™ ID v3.2 (Applied Biosystems, Foster City, CA) and FSS-i³ v4 (The Forensic Science Service^® DNA expert System Suite FSS-i³, Forensic Science Service, Birmingham, UK) with GeneScan^® Analysis v3.7/Genotyper^® v3.7 software (Applied Biosystems, Foster City, CA, USA) with manual review. We have shown that FaSTR DNA provides an alternative solution to automating DNA profile analysis and is appropriate for implementation into forensic laboratories. The FaSTR DNA system was demonstrated to be comparable in performance to that of GeneMapper™ ID v3.2 and superior to that of FSS-i³ v4 for the analysis of DNA profiles from crime samples.     

Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.     

A brief history of the formation of DNA databases in forensic science within Europe

The introduction of DNA analysis to forensic science brought with it a number of choices for analysis, not all of which were compatible. As laboratories throughout Europe were eager to use the new technology different systems became routine in different laboratories and consequently, there was no basis for the exchange of results. A period of co-operation then started in which a nucleus of forensic scientists agreed on an uniform system. This collaboration spread to incorporate most of the established forensic science laboratories in Europe and continued through two major changes in the technology. At each step agreement was reached on which systems to use. From the beginning it was realised that DNA databases would provide the criminal justice systems with an efficient way of crime solving and consequently some local databases were created. It was not until the introduction of the amplification technology linked to the analysis of short tandem repeats that a sufficiently sensitive and robust system was available for the formation of efficient and effective DNA databases. Comprehensive legislation enacted in the UK in 1995 enabled forensic scientists to set up the first national DNA database which would hold both personal DNA profiles together with results obtained from crime scenes. Other countries quickly followed but in some the legislation has severely restricted the amount and type of data which can be retained and, therefore, effectiveness of the databases is limited. The widespread use of commercially produced multiplex kits has produced a situation in which nearly all European laboratories are using compatible systems and there is, therefore, the potential for the introduction of a pan-European DNA database. However, the exchange of results between countries is hampered by the various legislations which currently exist.     

A fast analysis system for forensic DNA reference samples

On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a new analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA^® card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog.     

Successful extraction of human genomic DNA from serum and its application to forensic identification

We report here on the successful extraction of human genomic DNA from a serum sample in a forensic case. The extracted DNA was successfully used for the identification of remains presumably immersed for more than three weeks for which the only comparison sample was a 250-microL serum aliquot kept frozen in a laboratory. The analysis made it possible to identify a second victim as the daughter of the first.     

Polymorphism of structural genes of human mitochondrial DNA for forensic medical personal identification

An experimental study is described, which deals with polymorphism of nucleotide sequences of three structural genes of mitochondrial genome, i.e. of the 3d, small 4th, and 6th subunits of the NADN dehydrogenase complex (ND3, ND4L and ND6) sampled from Russian population. The genetic primary structure was analyzed in 63 unrelated individuals. The investigated locuses were shown to possess a pronounced polymorphism. A total of 19 polymorphic positions were detected in the ND3, ND4L and ND6 gene region within the studied sampling. Besides, a possibility is demonstrated in the paper that the mtDNA structural genes can be used as additional identification markers in the forensic experimental typing of the mtDNA control region.