Typing the 1.1 kb control region of human mitochondrial DNA in Japanese individuals

This study presents a reliable method that uses high-fidelity long-range PCR and optimized primers to assess polymorphism and to genotype human mitochondrial DNA (mtDNA). This method was used to analyze polymorphic sites in the human mtDNA control region, including hypervariable regions I, II, and III (HVI, HVII, and HVIII), from 124 unrelated Japanese individuals. In HVI, HVII, and HVIII, 80, 37, and 14 polymorphic sites were identified, respectively, excluding those in the homopolymeric cytosine stretch (C-stretch) regions. The region between HVI and HVII also contained 15 polymorphic sites. On the other hand, C-stretch length heteroplasmy in HVI or HVII was observed in 66 of 124 Japanese individuals (53%), which is much higher than in Caucasian populations. The variants in the C-stretch regions were characterized by counting the number of heteroplasmic peaks split from the single peak in homoplasmic sequences (i.e., 16244G and 16255G in HVI and 285G in HVII). Including the C-stretch length heteroplasmy, the 124 Japanese mtDNA samples were classified into 116 distinct haplotypes. The random match probability and the genetic diversity were estimated to be 0.95% and 0.998581, respectively, indicating that the method presented here has higher discrimination than the conventional method for mtDNA typing using HVI and HVII. [Correction added after publication 30 January 2007: in the preceding sentence random match probability and genetic diversity estimates were corrected from 0.95 and 0.998581%, respectively, to 0.95% and 0.998581, respectively.] The haplogroups and their frequencies observed in this study (i.e., D4; 13.7%, M7a1; 11.3%, D4a; 9.7% and M7b2; 8.9%) were similar to those observed in other studies of Japanese mtDNA polymorphism. The method described here is suitable for forensic applications, as shown by successful analysis of tissues from highly putrefied remains of an infant, which allowed maternal relationship to be determined via mtDNA haplotyping.     

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.     

Personal identification from human remains by mitochondrial DNA sequencing

The authors report four cases in which severely damaged human remains were identified by mitochondrial DNA (mtDNA) sequencing. Degraded DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for sequencing of two hypervariable regions, hypervariable region 1 and hypervariable region 2, of mitochondrial DNA. They also sequenced these regions of blood samples that were obtained from the presumptive mother or sister of the human remains. The sequencing results were compared with each other and with the Anderson's sequence. It was concluded from the sequence data that a lower part of a body in case 1 and some organs in case 2 were from the same woman, and a human head in case 3 and a female body in case 4 were from the relative of a presumptive mother and a sister, respectively.     

Determination of group affiliation of isolated spermatozoa after separation from squamous epithelium cells

Outlined in the paper are outlooks for determining a group affiliation of sperm in traces, left in material evidence, within forensic biological expertise. A technique is suggested to separate the spermatozoids from the vaginal epithelium cells--it has been used up to now in genetics alone. The method involves the available reagents and does not need any extra training. Its elaboration and introduction would resolve one of the most often-encountered issues set by the investigating office.     

Quantification of human mitochondrial DNA using synthesized DNA standards

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.     

Quantification of mitochondrial DNA in human blood cells using an automated detection system

The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.     

Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40–50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.     

Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40-50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.     

Sequencing of mitochondrial DNA and the problem of human specificity

When analysing trace materials and degraded DNA the issue of human specificity is highly important. Especially when it comes down to the analysis of mitochondrial DNA which is extremely susceptible to contamination authenticity is the main question. Therefore in the presented study mitochondrial primers were tested on their human specificity. In all cases it was possible to amplify DNA of animals with human mt-primers. These unintentional amplifications could only be decreased by choosing austere PCR parameters. The study implies the importance of comprehensive evaluation of primers, chemicals and PCR parameters.     

DNA Profiles from Fingerprint Lifts—Enhancing the Evidential Value of Fingermarks Through Successful DNA Typing

This study evaluated the compatibility of the most common enhancement methods and lifting techniques with DNA profiling. Emphasis is placed on modern lifting techniques (i.e., gelatin lifters and Isomark?) and historical fingerprint lifts for which limited research has been previously conducted. A total of 180 fingerprints were deposited on a glass surface, enhanced, lifted, and processed for DNA typing. DNA could be extracted and profiled for all the powders and lifts tested and from both groomed fingerprints and natural prints with no significant difference in the percentage of profile recovered. DNA profiles could also be obtained from historical fingerprint lifts (79.2% of 72 lifts) with one or more alleles detected. These results demonstrate the compatibility between different powder/lift combinations and DNA profiling therefore augmenting the evidential value of fingerprints in forensic casework.     

Extraction and analysis of human nuclear and mitochondrial DNA from electron beam irradiated envelopes

The United States Postal Service is considering methods such as electron beam irradiation to neutralize biological agents sent through the mail. While this is proven to reduce/eliminate pathogenic organisms, it may also degrade human genomic DNA and therefore hinder the ability to garner forensically informative genetic profiles. To determine the effects of electron beam irradiation on DNA typing, 16 white, standard letter-sized envelopes were licked. Half of the envelopes served as nonirradiated controls while the other half underwent irradiation at dosages sufficient to kill anthrax spores (29.3 and 51.6 kGy). Total cellular DNA was extracted from all envelopes; nuclear short tandem repeat loci, as well as the hypervariable region I from mitochondrial DNA, were amplified by means of the polymerase chain reaction. Short tandem repeat profiles and mitochondrial DNA sequence haplotypes were acquired on an ABI Prism 310 Genetic Analyzer platform. Analysis of data from irradiated samples revealed evidence of DNA degradation; however, the ability to construct full genetic profiles from both nuclear and mitochondrial DNA remained largely unaffected. The use of the polymerase chain reaction, coupled with florescent fragment analysis and mitochondrial DNA sequencing, should be considered to profile biological material from evidence enduring irradiation to inactivate infectious agents.     

Combined DNA Typing and Protein Identification from Unfired Brass Cartridges,,,

Biological evidence analysis from contact traces is adversely affected by low quantity and quality of DNA. Proteins in these samples contain potentially individualizing information and may be particularly important for difficult surfaces such as brass, where DNA may yield incomplete profiles. In this study, touched unfired brass cartridges were sampled using dry tape or wet swabs and analyzed by separating DNA and protein from the same collected material, thus producing both genomic and proteomic information. DNA recovery was similar for both collection methods, with tape yielding an average of 1.36 ± 1.87 ng and swabs, 1.34 ± 3.04 ng. Analysis by mass spectrometry identified 95 proteins, with the two collection methods showing no significant difference (p = 0.76) in the average number of collected proteins: 44.5 ± 10.9, (tape) versus 47.9 ± 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA evidence’, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such ‘blind-swabbing’ approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a ‘smart analysis’ single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male ‘assailant’ grabbing the wrist, neck or clothing from the female ‘victim’, or being in transient contact with bedding from the ‘victim’). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor ‘assailant’ DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.     

Quantification of human mitochondrial DNA in a real time PCR

Recently, a moderately priced machine for real-time quantitative PCR has become available, the Perkin Elmer 5700. The rapid and quantitative assay of mitochondrial DNA (mtDNA) copy number is potentially useful in a variety of molecular, evolutionary and forensic fields. Using this new tool, we have evaluated the precision and reliability of the real time PCR to quantify undeleted mitochondrial genome copy number, and to determine the frequency of an age-associated deletion of 4977 base pairs in length, in 42 human iliopsoas muscle DNA samples from persons of known age. We have evaluated the accuracy with which age can be predicted, knowing only the frequency of this common 4977 bp deletion, and derived a statistical formula which describes the confidence with which the 4977 bp frequency predicts age. The results indicate that the mutation frequency could be used to distinguish between tissue from young and old individuals. However in this data set, while there was considerable agreement of 4977 bp frequency among replicates from the same individual sample, there was substantial diversity of mean mutation frequency between individuals of the same or similar ages. The simplest interpretation of these results is that there are biological modifiers of 4977 bp frequency that are age-independent, which are potentially interesting but may limit the usefulness of this deletion frequency alone as a "molecular forensic clock."     

Critique of interpretation of high levels of heteroplasmy in the human mitochondrial DNA hypervariable region I from hair

The phenomenon known as heteroplasmy can be operationally observed in some human mitochondrial DNA (mtDNA) samples. Typically, heteroplasmy manifests itself in an individual presenting two mtDNA species that differ at a single base. Heteroplasmy at two, and even possibly three sites, also may occur, but at very low rates. A recent report (Grzybowski, 2000, see ref. [13]) suggests that much higher levels of mtDNA (point substitution) heteroplasmy can occur in hair. This observation is contrary to the experience of the forensic mtDNA community. There are several explanations for the unusual findings of high levels of heteroplasmy. First, the template quantities of DNA are approximately three orders of magnitude higher than required for mtDNA sequencing, and an excessive number of amplification cycles were used. Thus, the protocol used did not follow routine practices by the forensic community. Second, there are misidentifications and tabular errors that call into question the reliability of the findings. Third, by comparing the natural human mtDNA variation with a reference sample population with that observed in the heteroplasmy in hair study, the data are inconsistent with population genetic expectations. The observation of high levels of heteroplasmy may be due to contamination of the samples and/or possibly the amplification of nuclear pseudogenes. The results observed in the heteroplasmy in hair study do not apply to other methods of mtDNA analysis and cannot be used to question the reliability of the current forensic mtDNA practices.     

Typing of the locus DYS19 from DNA derived from fingernail clippings using PCR Concert rapid purification system

DNA extracted from the fingernails of female victims of a violent or aggressive act may assist in the identification of the male. Sometimes with the current autosomal STR loci, however, the victim's profile may mask the perpetrator's DNA profile or the perpetrator's DNA may be substantially lower in quantity than that of the victim's DNA. Thus, under these conditions, no characterization is possible. In this paper, an alternative DNA extraction procedure was employed, and the application of an STR locus residing on the Y chromosome DYS19 was typed to allow for genetic characterization of the perpetrator in such cases.     

Human and insect mitochondrial DNA analysis from maggots

During the course of our forensic investigations, we have encountered situations where it would have been useful to have evidence, other than direct contact between the two, for concluding that a carrion-fly maggot developed on a particular human victim. If a maggot collected during a death investigation did not develop on the victim, then its age is not relevant to estimating the postmortem interval. In this study we demonstrate that mitochondrial DNA (mtDNA) sequence data can be obtained from the dissected gut of a maggot that had fed on human tissue. These data can be used to identify both the human corpse upon which the maggot had been feeding and the species of the maggot itself.     

Length variation in HV2 of the human mitochondrial DNA control region.

Hair samples were typed from three individuals who exhibited length heteroplasmy in the homopolymeric cytosine stretches (C-stretch) in hypervariable region 2 (HV2). The study demonstrated that for different hairs within an individual, the HV2 C-stretch region can vary with respect to the number of cytosines and/or proportion of C-stretch length variants. Length heteroplasmy may occur regardless of the prominent length variant present in this region. Differences in the number of cytosines at the C-stretch region, or a variation in the relative amounts of heteroplasmic length variants, cannot be used to support an interpretation of exclusion.     

人骨骼肌线粒体DNA4977片段缺失与年龄的相关性调查

目的 探讨人骨骼肌组织中线粒体DNA 4977bp片段的缺失情况及其与年龄的相关性.方法 收集105例不同年龄死者的骨骼肌组织,抽提MDT,通过CPR、琼脂糖紫外凝胶成像等技术,测定不同年龄组线粒体DNA 4977bp片段的缺失情况.结果 在105例样品中,不同年龄组(0～9、10～19、20～29、30～39、40～49、50～59、60～69、70～79、80～89、90～99岁)mtDNA4977片段缺失的频率分别为:0、0、0.003%、0.011%、0.015%、0.033%、0.038%、0.062%、0.069%、0.091%.结论 人骨骼肌线粒体DNA 4977bp片段的缺失频率随着年龄的增长而增高,各年龄组之间具有显著差异(P《0.01).该缺失片段的检测对于软组织年龄推测具有一定指导意义.