Short tandem repeat (STR) genotyping of keratinised hair. Part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles

A feasibility study of short tandem repeat (STR) genotyping of telogen phase hairs in particular, and hair shaft in general, is presented. A number of extraction procedures in common use were investigated and the quantities of nuclear DNA (nuDNA) delivered were quantified via a real-time PCR assay. The extracts were subjected to two variations on AmpFlSTR Profiler Plus PCR amplification strategies (extended cycles, two rounds of PCR) and the genotypes compared. Nuclear DNA was found to persist in human hair shafts, albeit at very low levels. Full Profiler Plus profiles consistent with the hair donor were obtained from 100 mg hair shaft samples (bleached and unbleached). These were, however, mixed profiles, indicating low copy number (LCN) contamination in the extracts. Single telogen hair clubs and single hair shafts delivered partial profiles with usually only one allele of heterozygous loci. Telogen phase hairs yielded the same amount of nuDNA (and no more) as hair shafts (either anagen or telogen). Whether hair shafts dissolved or not in lysis buffer had no effect on either the quantitated yield of DNA or on the chance of obtaining a correct genotype. These results provide evidence that genomic DNA resides on the exterior of the hair shaft and we use this information to suggest an optimal procedure for nuDNA extraction from keratinised hair samples: soaking hairs in simple digestion buffers containing Tris-HCl, a salt and a chelating agent without prior cleaning of the hair shafts. It is proposed that cleaning removes most of the recoverable DNA. This procedure was applied to obtain genotypes from 3 cm hair shafts which matched reference profiles from the donors at up to 9 out of 10 AmpFlSTR Profiler Plus STR loci. When the genotyping success was measured by counting the number of matches between the two dominant alleles at each locus for each extract with the reference DNA profile of the hair donor, the success was found to be highly dependent on the donor. The number of matching alleles varied between not less than 10 for one donor to no more than two for another donor. These results may well be linked to the environmental experience of the hairs from each donor prior to removal.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

Deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs in Japanese.

The deoxyribonucleic acid (DNA) typing of human leukocyte antigen (HLA)-DQA1 from single hairs is described. HLA-DQA1 genotypes could be determined from single plucked hair roots. However, it was not easy to type HLA-DQA1 with hair shaft portions. Increase in the specimens of hair shaft portions (over 10 cm in length) to get sufficient DNA caused inhibition of polymerase chain reaction (PCR). Synthetic melanin as well as the one extracted from hairs inhibited the PCR of the genomic DNA template when added to the PCR reaction at the concentrations over than 15 ng/100 microL. Therefore, typability of hair shaft portions seems to depend on the delicate balance of the concentrations of DNA and the contaminated melanin in the final DNA extracts.     

The ultrastructure of tissue attached to telogen hair roots

Most tissues encountered in forensic biology laboratories have been previously characterized with electron microscopy due to their medical importance. Anagen hair root cells, epithelial cells, erythrocytes, neutrophils, osteocytes, and spermatozoa have received considerable research attention at the ultrastructural level. There is no literature indicating that cells attached to removed telogen hair roots have been characterized with transmission electron microscopy. Nonetheless, telogen hairs are a frequent submission item to forensic laboratories for DNA typing. The amount of tissue attached to a telogen hair root usually determines whether that hair is suitable for nuclear DNA typing methods or mitochondrial DNA typing methods. This study used transmission and scanning electron microscopy to characterize the tissues found in three commonly occurring telogen hair root forms. The tissues were found to consist of keratinized remnant follicle, nonnucleated epithelial cells, nucleated epithelial cells, and trichilemmal keratin. These findings were consistent with the known principles of hair follicle regression. The recognition of the root structures that contain these specific tissue types may assist in the DNA typing of telogen hairs inasmuch as the quality of tissue present may be more important than the amounts of tissue present.     

脱落毛发及毛干DNA的STR分型研究

目的探讨脱落毛发及毛干细胞核DNA提取、含量和STR分型问题。方法对脱落毛发或毛干进行DNA提取和定量,使用低扩增体系、增加循环次数和多次平行扩增等方法扩增DNA样本,采用叠加比较的方法分析STR分型。结果15cm脱落毛发样品DNA含量大于0．3ng的样品占52．8％,STR分型成功率为55．6％;15cm毛干样品DNA含量大于0．3ng的样品占30．6％,STR分型成功率为25％。结论采用增加循环次数、多次平行扩增等LCN—STR分型方法和Mini—STR试剂盒有助于脱落毛发及毛千的STR基因座分型获得。     

Evaluation and quantification of nuclear DNA from human telogen hairs

Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.     

Degeneration of Nuclei and Mitochondria in Human Hairs

Abstract:  It is generally accepted that nuclei degrade in developing hair shafts but the point at which such occurs has not been investigated. The fate of mitochondria in the keratinizing hair shaft has been less clear. This study uses transmission electron microscopy to investigate when nuclei and mitochondria are no longer visible in the developing hair shaft. Serial sections were obtained from anagen head hairs absent follicles in order to determine the sequence of degradation of nuclei and mitochondria in the hair shaft by starting at the root bulb and proceeding toward the hair tip. It was demonstrated that nuclei and mitochondria become invisible in the keratinizing hair shaft at about the same time. This was found to occur fairly early in the process at the level of the hair shaft where the hair cuticle becomes permanent.     

Proximal End Root Morphology Characteristics in Antemortem Anagen Head Hairs

The proximal end morphology of antemortem anagen head hair was compared with the characteristics documented to occur in postmortem hairs. Antemortem anagen and telogen head hairs (N = 967) were recovered following exposure to seven environments. Root morphology characteristics consistent with those reported in postmortem hairs were observed in 66 (14%) hairs exposed to a water, normal saline, outdoor soil, or indoor shower environment. Thirty‐three anagen hairs (7%) exhibited a root band at the proximal end. The mean distance from the root tip to the onset of the root band ranged from 0.23 to 0.7 mm, depending on the environment. The mean distance from the root tip to the onset of the root band was 0.46 mm, with a mean band length of 0.44 mm. The results illustrate the need to better characterize postmortem banding through quantitative measurements, including the range for root tip to band distance and the overall band length.     

Correlation of microscopic and mitochondrial DNA hair comparisons

Expert opinions regarding the microscopic comparison of human hairs have been accepted routinely in courts for decades. However, with the advent of mitochondrial DNA (mtDNA) sequencing, an assessment can be made of the association by microscopic hair comparisons in casework between a questioned hair and reference hairs from an individual. While each method can be used separately, the two analytical methods can be complementary and together can provide additional information regarding source association. Human hairs submitted to the FBI Laboratory for analysis between 1996 and 2000 were reviewed. Of 170 hair examinations, there were 80 microscopic associations; of these, only nine were excluded by mtDNA. Importantly, 66 hairs that were considered either unsuitable for microscopic examinations or yielded inconclusive microscopic associations provided mtDNA results. Only six hairs did not provide sufficient mtDNA, and only three yielded inconclusive results. Consistency was observed in exculpatory results with the two procedures. This study demonstrates the utility of microscopic hair examinations and the strength of combining microscopic analysis with mtDNA sequencing.     

Human hair histogenesis for the mitochondrial DNA forensic scientist.

Analysis of mitochondrial DNA (mtDNA) sequence from human hairs has proven to be a valuable complement to traditional hair comparison microscopy in forensic cases when nuclear DNA typing is not possible. However, while much is known about the specialties of hair biology and mtDNA sequence analysis, there has been little correlation of individual information. Hair microscopy and hair embryogenesis are subjects that are sometimes unfamiliar to the forensic DNA scientist. The continual growth and replacement of human hairs involves complex cellular transformation and regeneration events. In turn, the analysis of mtDNA sequence data can involve complex questions of interpretation (e.g., heteroplasmy and the sequence variation it may cause within an individual, or between related individuals. In this paper we review the details of hair developmental histology, including the migration of mitochondria in the growing hair, and the related interpretation issues regarding the analysis of mtDNA data in hair. Macroscopic and microscopic hair specimen classifications are provided as a possible guide to help forensic scientists better associate mtDNA sequence heteroplasmy data with the physical characteristics of a hair. These same hair specimen classifications may also be useful when evaluating the relative success in sequencing different types and/or forms of human hairs. The ultimate goal of this review is to bring the hair microscopist and forensic DNA scientist closer together, as the use of mtDNA sequence analysis continues to expand.     

Fungal tunneling of hair from a buried body.

Tunnels produced in human head hair by fungal hyphae were examined with a light microscope and with a scanning electron microscope. The tunnels had small diameters and exhibited minimal branching. The use of a backscattered electron detector facilitated the locating of the openings of the tunnels in the surfaces of the hairs. In the backscattered electron image, tunnel openings appeared as dark spots. The tunneling hyphae did not show a preference for a particular location for entering the shaft of the hair. Some hyphae penetrated under the free edges of the cuticular scales, while others burrowed through the surfaces of the scales.     

The ABO blood grouping of a minute hair sample by the immunohistochemical technique

The unlabeled antibody (PAP) immunoperoxidase technique was applied to the ABO blood grouping of human scalp hairs. Hair samples were subjected to longitudinal- or cross-sectioning, thus obtaining suitable samples for subsequent immunostaining. The immunostaining was carried out using rabbit anti-A and anti-B sera as the primary antibodies. With this technique, the group-specific staining which is revealed as a dark brown precipitate was clearly observed within the medullae of the hair shaft, and depending on the presence or absence of these precipitates, respective blood groups of unknown hair samples were determined. At the hair root, on the other hand, positive stainings were observed not only in medullary cells but also in some cortical cells of the keratogenous zone. From the present study, it can be safely said that this technique is of practical use for the ABO blood grouping from a minute (less than 3 mm) hair sample.     

Optimising direct PCR from anagen hair samples

Anagen hairs are in the active growth phase, and when forcefully removed, may contain an intact root or sheathing. The hair root or sheathing is a source of nucleic DNA and can be amplified using direct PCR. Human identification STR kits are optimised to a small range of input DNA for PCR. Anagen hairs are unable to be quantified prior to amplification and can exhibit characteristics of an over-loaded DNA sample when analysed. The aim of this study was to optimise direct PCR for anagen hair sampling. Two separate modifications to the downstream processes were carried out in order to determine the most effective method at minimising PCR artefacts. Decreasing the cycle number from the standard 29 cycles to 27 cycles when using the NGM™ kit displayed the best results for this method. However, decreasing the cycle number may increase allelic drop-out and would be costly for laboratories to perform an in-house validation. Diluting the PCR product during electrophoresis analysis minimises the effects of PCR artefacts in the same way decreasing the cycle number does. Diluting the PCR product is the most cost-effective method and does not increase the chance of allelic drop-out.     

The visualization and quantification of cell nuclei in telogen hair roots by fluorescence microscopy, as a pre-DNA analysis assessment

Although nuclear DNA-profiling of human hairs is a well-known technique in forensic investigations, its success rate is quite low. Because the extracted nuclear DNA (nuDNA) is scarce and often degraded, a simple and effective method was developed to estimate the number of cell nuclei in telogen roots. DAPI, a fluorescent, non-destructive DNA-stain, allows visualizing nuclear DNA and does not interfere with subsequent PCR analyses. After staining 3242 roots from 27 volunteers and subsequent STR-profiling of a selection of roots, we show that the amount of analysable nuDNA can be predicted. This screening method allows the genetic laboratory to analyse only the most promising hair roots.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

Microscopical discrimination of twins' head hair

Twin populations are ideal for studying human variation; a study of twin's hair, therefore, provided a better understanding of the value of hair comparisons. Duplicate head hair samples from 17 pairs of twins and one set of identical triplets were compared in a verified blind study. In addition to the direct comparison of all twins, random samples of two or three hairs were compared with randomly selected groups of known samples in a second blind study, to better simulate an ordinary forensic science case. Features commonly used by forensic hair examiners were adequate to distinguish hair samples from each twin from all other samples, illustrating the power of microscopical comparison when numerous questioned hairs are available in evidence. When two or three hairs were compared with randomly selected known samples, several were indistinguishable from hair samples other than the true source, proving once again that a human hair can never be associated with one person to the exclusion of all others.     

Characterization of Mitochondrial DNA Sequence Heteroplasmy in Blood Tissue and Hair as a Function of Hair Morphology*,†,‡

Abstract: This study characterizes mitochondrial DNA (mtDNA) sequence heteroplasmy in blood tissue and hair as a function of hair morphology. Bloodstains (127 individuals) and head hairs (128 individuals) were typed using the mtDNA LINEAR ARRAY? assay. A total of 1589 hairs were interpreted: 1478 (93%) were homoplasmic and 111 (7%) exhibited heteroplasmy at one or more positions. Seventy‐one percent (82/116) of individuals were homoplasmic, whereas 29% (34/116) exhibited heteroplasmy in at least one hair. The results demonstrate intra‐ and inter‐tissue differences in heteroplasmy within individuals. Sequence heteroplasmy among hairs from each individual varied from 0 to 90%; the frequency does not differ significantly with population group, cosmetic treatment, age, gender, medulla morphology, region of the scalp, hair growth phase, or, when comparing living and deceased donors. However, the results support a correlation between heteroplasmy and hair pigmentation; typically, lighter‐pigmented hairs exhibit a higher incidence of sequence heteroplasmy compared to darker hairs.     

单根毛发角蛋白的分离与分析

本文用单根毛发(1～5cm)进行微量化角蛋白 SDS-梯度聚丙烯酰胺凝胶电泳分析,并对人与5种不同种属的动物毛发角蛋白进行了比较研究。结果表明,人与动物毛发角蛋白组分有明显差异,主要区别在于低分子量区域;用 N-(3-芘)马来酰胺标记毛发角蛋白巯基,进一步确定了毛发角蛋白中低硫蛋白位于45,000～52,000分子量范围,高硫蛋白位于<18,500分子量范围。人与动物毛发角蛋白的主要差异是高硫蛋白。作者认为,本实验方法对法医学毛发鉴别有一定的实用意义。     

Resistance of degraded hair shafts to contaminant DNA

We have investigated the susceptibility of degraded human hair shaft samples to contamination by exogenous sources of DNA, including blood, saliva, skin cells, and purified DNA. The results indicate that on the whole hair shafts are either largely resistant to penetration by contaminant DNA, or extremely easy to successfully decontaminate. This pertains to samples that are both morphologically and biochemically degraded. We suggest that this resistance to the incorporation of contaminant DNA relates to the hydrophobic and impermeable nature of the keratin structures forming the hair shaft. Therefore, hair samples represent an important and underestimated source of DNA in both forensic and ancient DNA studies.     

The persistence of human scalp hair on clothing fabrics

This study reports the persistence behaviour of human scalp hairs under a number of different circumstances. The effects of artificial dyeing of hairs, the presence or absence of roots and different types of fabrics on the persistence of hair on a variety of garments were investigated. The garments were made from cotton, polycotton, cotton/acrylic, polyester and wool. The results indicated that neither artificial dyes nor the presence or absence of roots had statistically significant effects on the persistence of hair. In contrast, the type of fabric had a major impact and it was found that, generally, hairs persist longer on rougher fabrics. The rate of loss of hairs from non-woollen fabrics during normal wear was found to follow an exponential decay curve. In contrast, the rate of loss from the woollen garments was quite linear, indicating a constant, even loss, and thus suggests that a different process is involved in the persistence of hairs on woollen garments from that on non-woollen garments. The speed at which hair was lost from fabrics decreased in the order polyester, cotton/acrylic, polycotton, cotton, smooth wool, rough wool, so that wool gives the best chance of recovering samples of hair. Due to the uniqueness of each case, it is advised that caution be used when making any interpretations and before drawing any conclusions.