Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Touch DNA has become increasingly important evidence in todays' forensic casework. However, due to its invisible nature and typically minute amounts of DNA, the collection of biological material from touched objects remains a particular challenge that underscores the importance of the best collection methods for maximum recovery efficiency. So far, swabs moistened with water are often utilized in forensic crime scene investigations for touch DNA sampling, even though an aqueous solution provokes osmosis, endangering the cell's integrity. The aim of the research presented here was to systematically determine whether DNA recovery from touched glass items can be significantly increased by varying swabbing solutions and volumes compared with water-moistened swabs and dry swabbing. A second objective was to investigate the possible effects of storage of swab solutions prior to genetic analysis on DNA yield and profile quality when stored for 3 and 12 months, as is often the case with crime scene samples. Overall, the results indicate that adapting volumes of the sampling solutions had no significant effect on DNA yield, while the detergent-based solutions performed better than water and dry removal, with the SDS reagent yielding statistically significant results. Further, stored samples showed an increase in degradation indices for all solutions tested, but no deterioration in DNA content and profile quality, allowing for unrestricted processing of touch DNA samples stored for at least 12 months. One further finding was a strong intraindividual change in DNA amounts observed over the 23 deposition days which may be related to the donor's menstrual cycle.     

Tissue storage solution for preservation and transfer of forensic specimen in high ambient-temperature

Storage of tissue samples in high ambient-temperature can affect the quality of forensic evidence. Experiments were conducted to investigate the potential use of 3 tissue storage solutions for the preservation and transfer of forensic specimen in high ambient temperature conditions, i.e., DMSO, Longmire’s buffer, and trehalose solution. Results showed that DNA in tissue was best preserved in DMSO buffer. Samples preserved in Longmire’s buffer gave DNA analysis results for temperatures up to 60 °C, however, amplification between replications were not reproducible. For those tissue samples preserved in trehalose solution, DNA markers larger than 300 bp were absent, and irreproducible amplification results were detected at a higher level when the storage temperature increased, and storage time was over 2 weeks. Tissue storage condition at high temperature over 1 week is not recommended. Experimental results here provided an alternative collection and preservation method for tissue samples at ambient temperature (without cold-storage) for subsequent DNA analysis. These can potentially be implemented in forensic biological evidence collection, preservation and transfer in hot climates.     

Storage of Second World War bone samples: Bone fragments versus bone powder

Bone samples may yield low-quality and low-quantity DNA and duplicated analyses of different genetic markers have to be performed for identification of missing persons. Mostly no DNA extract is left after analyses and efficient storage of bones is needed to ensure the stability of the sample over time for retesting using new markers and new technologies. Usually not all of the bone powder prepared in grinder is used for extraction and rest can be stored for future analyses. After molecular genetic analyses of 88 victims of Second World War (WWII) Konfin I mass grave in Slovenia (performed in 2009), fragments of femurs and bone powder that were left were stored at -20 °C. Some authors reported that long-term storage of powder results in the reduction of DNA preservation and its degradation (even at low temperature), explained by an increase in oxidative damage as a result of the enormous increase in exposed surface area. Consequently, grinding of bones as shortly prior to DNA extraction was recommended. The goal of our study was to explore the difference in DNA yield between bone fragment and bone powder frozen for 10 years. 57 WWII femurs were examined and DNA extracted from each of them using bone fragment (piece sampled next to the one used in 2009) and bone powder obtained in 2009, both taken out of freezer after 10 years of storage. Half gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen) and quantified with PowerQuant kit (Promega). Statistical analysis showed significant difference at the 0.05 level in DNA yield comparing fragments of bones and bone powder stored at -20 °C for 10 years. The results show there is more DNA stored in the bone powder than in the bone fragments. Because of time - consuming powdering procedure we recommend to store not only the fragment of the bone, but obtained bone powder as well.     

Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent

DNA extraction from trace samples or noninvasively collected samples often results in the recovery of low concentration solutions of DNA that are prone to DNA degradation or other loss. Because of the difficulty in obtaining such samples, and their potentially high value in wildlife and forensic studies, it is critical that optimal methods are employed for their long-term storage. We assessed the amplification yield of samples kept under different storage conditions with the addition of potential preserving agents. We stored dilutions of known concentration human placental DNA, and gorilla fecal DNA, under four conditions (+4 degrees C, -20 degrees C, -80 degrees C, dry at room temperature), and with three additives (Tris EDTA (TE) buffer, Hind III digested Lambda DNA, trehalose). The effectiveness of the treatment methods was tested at regular intervals using qPCR to assess the quantity of amplifiable DNA, and a PCR assay of a larger 757 bp fragment to evaluate the quality of that remaining DNA. The highest quantity of DNA remained in samples stored at -80 degrees C, regardless of storage additives, and those dried at room temperature in the presence of trehalose. Surprisingly, DNA quality was best preserved in the presence of trehalose, either dried or at -80 degrees C; significant quality loss occurred with -20 degrees C and +4 degrees C storage.     

Simplified field preservation of tissues for subsequent DNA analyses

Successful DNA-based identification of mass disaster victims depends on acquiring tissues that are not highly degraded. In this study, multiple protocols for field preservation of tissues for later DNA analysis were tested. Skin and muscle samples were collected from decaying pig carcasses. Tissues were preserved using cold storage, desiccation, or room temperature storage in preservative solutions for up to 6 months. DNA quality was assessed through amplification of successively larger segments of nuclear DNA. Solution-based storage, including a DMSO/NaCl/EDTA mixture, alcohols, and RNAlater preserved DNA of the highest quality, refrigeration was intermediate, and desiccation was least effective. Tissue type and extent of decomposition significantly affected stored DNA quality. Overall, the results indicate that any tissue preservation attempt is far superior to delaying or forgoing preservation efforts, and that simple, inexpensive methods can be highly effective in preserving DNA, thus should be initiated as quickly as possible.     

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.     

Comparison of DNA yield after long-term storage of Second World War bone samples

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.     

Assessment of PowerPlex® Fusion 5C’s ability to type degraded DNA

DNA samples collected at crime scenes are often degraded so this research focused on the ability of the Promega PowerPlex® Fusion 5C amplification kit to type both naturally and artificially degraded DNA.DNA was degraded naturally by placing equal volumes of blood on white fabric that was stored either inside, outside in a shaded area, or outside in direct sunlight. Samples were then collected every 10 days for 60 days and the DNA extracted (QIAamp® DNA Investigator). Artificially degraded samples were created by exposing extracted DNA to either UV light or 95 °C heat for varying times. DNA was also degraded artificially by placing blood samples into a 50% bleach solution for varying times prior to extraction.Following sample treatment, standard forensic DNA analysis was performed including quantification (Investigator® Quantiplex) and amplification (PowerPlex® Fusion 5C). Separation and detection were performed on an ABI 3130xl capillary electrophoresis unit and analysis was performed using GeneMapper ID v3.2.1.While the time and shade samples showed similar amounts of degradation, the samples exposed to direct sun showed more degradation. The artificially degraded samples showed more signs of degradation such as reduced overall peak height and peak height imbalance at heterozygous loci. There were also some cases where an allele that was known to be in the profile exhibited total dropout. Although there were some instances of both allelic dropout and heterozygote peak imbalance, PowerPlex® Fusion was able to reliably type degraded DNA as all alleles detected were consistent with the known donor profile. The results show that PowerPlex® Fusion is a robust kit capable of handling forensically challenged samples.     

激光显微捕获技术用于尿液脱落细胞DNA检测

目的采用激光显微捕获技术（LCM）捕获尿液脱落细胞,并进行STR分型。方法收集10份健康成人尿液样本,根据储存时间分组,其中新鲜尿液组（≤24h）分别采用Chelex-100及LCM联合DNA IQTM提取法提取DNA,储存尿液组（〉24h）再分为4℃组和室温组,分别在4～30d内不同时间点采用LCM联合DNA IQTM提取法提取DNA;各组提取的模板DNA进行扩增及SRT分型检验。结果新鲜尿液组采用LCM联合DNA IQTM提取法提取DNA,所有样本均可检出全部基因座（16个）,采用Chelex-100法则在部分基因座上出现等位基因丢失、非特异性扩增、峰值低等现象;4℃储存10d和室温储存4d以内的尿液经检验可明确判读12个以上基因座,4℃20～30d及室温7d,可检出7个以上基因座。结论 LCM技术可用于尿液检材的DNA分型检验,且检材应尽可能4℃保存并尽快检验。     

The effect of freezing,thawing and long-term storage on forensic DNA extracts

Following forensic DNA profiling (extraction, quantification and STR typing) the remaining extract is generally stored frozen. Our routine at the Swedish National Forensic Centre is to immediately after analysis freeze the sample. If a subsequent reanalysis is needed the sample is thawed and then refrozen. In this study the effects of freezing and thawing as well as long-term storage of DNA extracts in refrigerator or freezer have been investigated. The following sample types were extracted: two levels of blood and saliva, saliva on cigarette filter paper, saliva on cotton swabs and a combination of saliva and semen to mimic samples from sexual assaults. All extraction methods used were Chelex-based, DNA quantification was performed using PowerQuant System and STR profiling with PowerPlex ESX 16 Fast System. The study was divided into three parts: 1) freezing and thawing the extracts up to ten times, 2) storage in refrigerator or freezer up to four weeks and 3) long-term storage in refrigerator or freezer for 3, 6, 9, 12 and 35 months. Generally, the quantification and STR typing results show no indication of degradation after repeated freezing and thawing or long-term storage in refrigerator or freezer.     

Evaluation of direct PCR for routine DNA profiling of non-decomposed deceased individuals

Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow.     

The effect of temperature on Y-chromosome detection in blood stains

The present investigation is an extension of an earlier work on the effect of various temperatures on Y-chromosome detection. The deteriorating effect of storage at 53 °C was again demonstrated. Liquid blood samples stored at 5 °C were better preserved with regard to Y-chromosomes than those stored at room temperature. This was in conflict with the earlier results on blood stains. New experiments were therefore performed. The results varied. It is suggested that this variation was due to low temperatures having two contrary effects on the results: the well-known preserving effect on biological material and a previously described constricting effect on the heterochromatic areas of the chromosomes making Y-body detection more difficult.     

Effect of storage conditions on restriction fragment length polymorphism (RFLP) analysis of deoxyribonucleic acid (DNA) bound to positively charged nylon membranes.

The ability to generate an autoradiogram from deoxyribonucleic acid (DNA) immobilized on a positively charged nylon membrane could be compromised by the storage conditions of the membrane. HaeIII-digested human DNA was size fractionated and transferred to two types of positively charged nylon membranes. The membranes were stored at -20 degrees C, 4 degrees C, and ambient temperature and humidity for times ranging from 1 day to 13 weeks, then hybridized to variable number of tandem repeat (VNTR) probes to examine the effect of the storage conditions on the membrane-bound DNAs. It was shown that such membranes could be successfully hybridized and rehybridized if they were stored at -20 or 4 degrees C, but storage under ambient conditions reduced or eliminated the likelihood of successful hybridization.     

Evaluation and usefulness of reverse dot blot DNA-PolyMarker typing in forensic case work

Experiments were performed to evaluate the Amplitype PolyMarker DNA typing system for application to forensic casework. DNA extraction using chelex was compared with phenol-chloroform extraction for various biological materials including postmortem blood, blood samples used for alcohol quantification, fresh urine, envelopes and cigarette butts. Different amounts of genomic DNA were amplified to test the sensitivity of the Amplitype PM. Mixed samples of two different bloods were typed to determine the dilution at which mixtures could be detected. Different storage conditions were evaluated using urine samples. Postmortem blood samples were typed during 4 months to determine the effects of natural degradation. A population sample of 105 unrelated individuals from South-West Switzerland was analyzed and the genotype frequencies were compared with those reported by others. Finally, practical usefulness of the Amplitype PM system is illustrated by analysing casework samples. The results of this validation proved the great usefulness and sensitivity of the Amplitype PM system using the appropriate extraction and typing method. However, mixed samples had to be interpreted with caution owing to the possibility of non-specific alleles with stored material such as urine and postmortem blood.     

DNA IQ^TM SYSTEM在疑难指甲DNA检验中的应用

目的探索DNA IQTMSYSTEM在疑难指甲DNA提取中的应用。方法 15份疑难指甲采用Chelex方法检验没有成功获得STR分型图谱,采用DNA IQTMSYSTEM提取法并纯化,采用Identifiler PLUS试剂盒进行复合扩增,产物经ABI3130XL型DNA基因分析仪检测。结果成功获得15例疑难指甲的STR基因座DNA分型。结论 DNA IQTMSYSTEM方法能快速、有效提取疑难指甲DNA进行STR分型。     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions

Identifying vaginal secretions attaching or adhering to a suspect’s belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample.In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.     

Development of a forensic DNA phenotyping panel using massive parallel sequencing

The HIrisPlex-S system, targeting a total of 41 SNPs, allows the simultaneous eye, hair and skin color prediction from DNA. In the present study, we developed a massive parallel sequencing (MPS) multiplex assay in order to genotype all the HIrisPlex-S markers in degraded casework samples. PCR amplicons sizes of target regions were kept below 180 bp, in order to allow analysis of degraded DNA samples. Individuals with known phenotype, artificially degraded DNA samples and a set of 2800M control DNA dilutions were sequenced on a Ion PGM System, in order to evaluate the concordance testing results and the forensic suitability of this 41-plex MPS assay. Full and reliable profiles could be obtained with 0.1 ng of input DNA. The increment of the number of PCR cycles results in improvement of sensitivity or in typing results but an increase of artifacts were also observed.     

Bioinformatics and genetic privacy: The impact of the Personal Data Protection Act 2010

Bioinformatics refers to the practise of creation and management of genetic data using computational and statistical techniques. In Malaysia, data obtained from genomic studies, particularly for the purpose of disease identification produces a tremendous amount of information related to molecular biology. These data are created from DNA samples obtained from diagnostic and research purposes in genomic research institutes in Malaysia. As these data are processed, stored, managed and profiled using computer applications, an issue arises as to whether the principles of personal data privacy would be applicable to these activities. This paper commences with an illustration of the salient features of the Personal Data Protection Act 2010. The second part analyses the impact of the newly passed Personal Data Protection Act 2010 on the collection of DNA sample, the processing of data obtained from it and the profiling of such data. The third part of the paper considers whether the various personal data protection principles are applicable to the act of DNA profiling and the creation of bioinformatics.     

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5 mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.