Mitochondrial DNA typing screens with control region and coding region SNPs

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.     

A simplified method for mitochondrial DNA extraction from head hair shafts

DNA isolation from hair shafts can involve a number of steps, each of which adds time to the procedure and increases the risk of contamination. A simple alkaline digestion procedure that directly dissolves hairs was developed and compared with a widely used glass grinding/organic extraction method, using samples collected from 30 volunteers with varying population ancestries, hair colors, and hair treatments. A 203 bp mtDNA product could be amplified from 90% of samples extracted by alkaline digestion and 73% of hairs extracted by glass grinding. DNA obtained from alkaline digested hair generated equal or greater amplification success for virtually all criteria examined, and mtDNA sequences matched buccal control sequences in all cases. The two methods were similar in DNA yield (amplification success at template dilution) and quality of DNA obtained (amplicon length). Alkaline digestion of hair shafts required 6-7 h to complete, compared to 22-24 h for glass grinding, and proved a less laborious yet equally robust method for mtDNA extraction.     

Comparison of two methods for isolating DNA from human skeletal remains for STR analysis

Abstract: The quality and efficiency of a standard organic DNA isolation method and a silica‐based method using the QIAGEN Blood Maxi Kit were compared to obtain human DNA and short tandem repeats (STRs) profiles from 39 exhumed bone samples for paternity testing. DNA samples were quantified by real‐time PCR, and STR profiles were obtained using the AmpFlSTR^® Identifiler^® PCR amplification kit. Overall, the silica‐based method recovered less DNA ranging from 0 to 147.7 ng/g (average 7.57 ng/g, median = 1.3 ng/g) than did the organic method ranging from 0 to 605 ng/g (average 44.27 ng/g, median = 5.8 ng/g). Complete profiles (16/16 loci tested) were obtained from 37/39 samples (95%) using the organic method and from 9/39 samples (23%) with the silica‐based method. Compared with a standard organic DNA isolation method, our results indicate that the published silica‐based method does not improve neither the quality nor the quantity of DNA for STR profiling.     

Differentiation of Morphologically Similar Human Head Hairs from Two Demographically Similar Individuals Using Amino Acid Ratios,

Human hair is frequently encountered as forensic evidence and can contribute valuable information to investigators. Conventional forensic hair analyses include microscopic hair comparison (MHC) and DNA analysis. However, MHC is not supported by statistics and DNA analysis cannot always be performed. Recent studies have demonstrated that evaluation of differences in the hair proteins may offer an alternate method to these analyses. In this study, an evaluation of the amino acids present in hair was investigated as an approach to differentiate morphologically indistinguishable hair samples from two demographically similar individuals. Proteins in the hair were digested using hydrochloric acid, and the resulting amino acids were derivatized with N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) for analysis using gas chromatography-mass spectrometry (GC-MS). Eight derivatized amino acids were detected and quantified relative to an internal standard, L-norvaline, and used to construct twenty-eight amino acid ratios. Hair samples were collected from four areas of the head on various days over the course of one month, and no significant differences in amino acid ratios (p-value > 0.05) were observed among the areas of the head, and the ratios were consistent over the time period of this study. Additionally, fifteen of these amino acid ratios were found to be significantly different between the two individuals when compared using a two-sample t-test (p-value ≤ 0.05). These data indicate that amino acid analysis was able to differentiate two morphologically similar hair samples from different individuals and demonstrates the applicability of this method to distinguish similar hair samples when DNA analysis cannot be performed.     

Efficiency of DNA IQ System in recovering semen DNA from cotton swabs

With the aim to asses the efficiency of the DNA IQ System in the recovery of DNA from semen samples, cotton swabs were prepared from 1/5 serial dilutions of semen. Each swab was fractionated in four equivalent quarters and the DNA was further extracted following the differential lysis protocol. The recovered DNA was quantified by means of real time PCR and the average DNA yield was used to compare results. Direct extractions of equivalent aliquots of each semen dilution were used as reference samples. Even though a high percentage of the starting material was lost during the process of transfer to/recover from the solid support, our experimental results demonstrated that the DNA IQ system was able to detect around 10³ sperm cells in the starting material, enabling to obtain a complete DNA profile with AmpFl STR IdentiFiler PCR Amplification Kit (Applied Biosystems).     

Validation of the AMPFℓSTR® MiniFilerTM PCR Amplification Kit for Use in Forensic Casework*

Abstract: The AmpF?STR^® MiniFiler^TM PCR Amplification Kit is designed to genotype degraded and/or inhibited DNA samples when the AmpF?STR^® Identifiler^TM PCR Amplification Kit is incapable of generating a complete genetic profile. Validation experiments, following the SWGDAM guidelines, were designed to evaluate the performance of MiniFiler. Data obtained demonstrated that MiniFiler, when used in conjunction with Identifiler, provided an increased ability to obtain genetic profiles from challenged samples. The optimum template range was found to be between 0.2 and 0.6 ng, with 0.3 ng yielding the best results. Full concordance was achieved between the MiniFiler kit and Identifiler kit except in a single case of a null allele at locus D21S11. Numerous instances of severe heterozygous peak imbalance (<50%) were observed in single source samples amplified within the optimum range of input DNA suggesting that caution be taken when attempting to deduce component genotypes in a mixture.     

Development of a real-time method to detect DNA degradation in forensic samples

Abstract: Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real‐time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples.     

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400 μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpF?STR^® SGM Plus^® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.     

Analysis of forensic samples in Banco Nacional de Datos Genéticos

Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye^® Terminator v 1.1 Kit and analyzed in ABIPRISM^® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler^®, Minifiler^® and YFiler^® Kit (AB) and analyzed in ABI PRISM^® 3100 Genetic Analyzer and ABI PRISM^® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.     

Internal Validation of Human Mitochondrial DNA Quantification Using Real‐Time PCR

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real‐time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/μL or approximately six human mtDNA copies/μL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re‐purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.     

Removing and identifying drug contamination in the analysis of human hair

The procedure used in this laboratory for removing and identifying contamination of hair specimens with drugs is demonstrated by its application to hair contaminated by various experimental models. The models include soaking; coating with drug followed by sweat conditions for 6 h; and soaking in a very high concentration of cocaine followed by storage and multiple shampoo treatments. A multi-part wash procedure along with a wash criterion is applied to all samples containing drug above the cutoff. The failure of the wash criterion is a signal that the sample may be positive due to contamination rather than use, and in the absence of other over-riding evidence, the sample would be considered to be negative for drug use. This Wash Criterion has also been tested with hair from subjects demonstrated to be drug users by one or more drug-positive urines; in these studies, all hair samples from demonstrated users passed the Wash Criterion test.     

MtSNP typing before mtDNA sequencing: Why do it?

Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.     

Mitochondrial DNA sequencing of human hair shafts stored for long time

Mitochondrial DNA (mtDNA) sequencing is commonly used for forensic genetic identification of relation and personality identification based on analysis of tooth and skeletal rudiments. We demonstrated the possibility of DNA extraction and subsequent enzymatic amplification of fragments of a hypervariable segment I of mtDNA control region from hair shafts after long storage (up to 75 years). Shed hairs are the most common biological material evidence in forensic investigations. Low content of DNA and its possible degradation in hair shafts without bulbs may cause artifacts in polymerase chain reaction. However comparative analysis of amplified nucleotide sequences of amplified fragments from hair stored for 75 years was identical to the sequence from hairs cut immediately before experiment. This indicates high quality of the resultant matrices, stability of results, and hence, the possibility of using DNA extracted from hair shafts without bulbs stored for a long time for expert genetic analysis. Theoretical and methodological prospects of using mtDNA polymorphism analysis for forensic expert evaluations are discussed.     

用引物Y_3,Y_4和DNA扩增技术作血液(痕)和毛根性别鉴定的研究

作者用引物Y_3、Y_4和DNA聚合酶链式反应(PCR)作微量人类血液(痕)和毛根的性别鉴定。扩增的靶序列位于Y染色体DNA特异3.4kb重复序列中,扩增产物为460bp。检材用量为:新鲜血液0.5μl、血痕纱纤维1mm、毛根单个。20例保存4个月的血痕与2例保存6年半的血痕性别判定结果均正确,无性别记载的保存9～11年的3例血痕显现了清晰的460bpY特异DNA扩增带。15例保存20天的自然脱落毛根性别判定结果均正确。本法省略了检材处理中的酚-氯仿抽提DNA等纯化步骤,既简化了实验操作,又减少了检验过程中外源DNA的污染机会和样品DNA的损耗,使这一性别鉴定方法更符合法医学实践的需要。     

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples*†

Abstract: Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C‐stretch and CA repeat. To observe which CA “alleles” were present in each tissue, PCR products were cloned and re‐sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.     

Application of mtDNA SNP analysis in forensic casework

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.     

A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth

DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler? BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique.     

Evaluation of five DNA extraction systems for recovery of DNA from bone

Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch^® gDNA Plant Kit (Life Technologies), DNA IQ^TM System Kit (Promega), DNeasy^® Blood & Tissue Kit (Qiagen) and PrepFiler^® BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq^® qPCR Master Mix (Promega) using an Applied Biosystems^® 7500 Real-Time PCR System and the extracts were amplified using an in-house multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch^® gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition.     

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.     

Heteroplasmy in hair: differences among hair and blood from the same individuals are still a matter of debate

The analysis of mitochondrial DNA (mtDNA) is a useful tool in forensic cases when sample contents too little or degraded nuclear DNA to genotype by autosomal short tandem repeat (STR) loci, but it is especially useful when the only forensic evidence is a hair shaft. Several authors have related differences in mtDNA from different tissues within the same individual, with high frequency of heteroplasmic variants in hair, as also in some other tissues. Is still a matter of debate how the differences influence the interpretation forensic protocols. One difference between two samples supposed to be originated from the same individual are related to an inconclusive result, but depending on the tissue and the position of the difference it should have a different interpretation, based on mutation-rate heterogeneity of mtDNA. In order to investigate it differences in the mtDNA control region from hair shafts and blood in our population, sequences from the hypervariable regions 1 and 2 (HV1 and HV2) from 100 Brazilian unrelated individuals were compared. The frequency of point heteroplasmy observed in hair was 10.5% by sequencing. Our study confirms the results related by other authors that concluded that small differences within tissues should be interpreted with caution especially when analyzing hair samples.