Sufficient Amount Of DNA Research Articles

Biological traces at the crime scene

Biological traces found at the crime scene are of human, animal and plant origin. Among them the most commonly present are of human origin - hair, blood, sperm, saliva, sweat and tears, but also nails, teeth and bone bits. When considering biological traces as evidence, we distinguish incontestable and debatable traces. Incontestable samples are taken from suspects, alive victims and from all other people that may have been in contact with the traces or objects at the crime scene. Debatable traces are found at the scene of events, on objects or people associated with a venue or criminal offense, whose identity and role in the event is known. When taking biological traces, it is necessary to comply with the anti-contamination and safety rules regarding handling with biological material, including self-protection measures. The goal of processing biological traces that are usable as court evidence is to provide a sufficient amount of DNA, which can be used in the individualization of each biological trace. However, in addition to this analytical step by which evidence is undoubtedly drawn from the trace, biological traces can be treated with some simpler, cheaper and less reliable methods. In this review article, we also discussed the judicial significance of sperm traces. Sperm is often analyzed for crimes impingeing on sexual freedom. Hair samples can be used for toxicological analysis and to connect perpetrators to an event or victim.

Early fetal sexing in the rhinoceros by detection of male-specific genes in maternal serum.

Genetic sexing of animals with long gestation time benefits the management of captive populations. Here, X and Y chromosome-specific primers, based on equine gene sequencing data, were developed and tested on captive rhinoceroses (10 males, 20 females) representing four species (Diceros bicornis, Certaotherium simum simum, Rhinoceros unicornis, and Dicerorhinus sumatrensis). The Y chromosome-specific primer set targeted SRY (Sex-determining region Y), and amplified a 177-bp product following PCR of DNA extracted from males, but not females, of all species. A primer set based on the equine AMEL (Amelogenin) gene resulted in a 232-bp product following PCR of all rhinoceros species. These gene-specific primer sets were then evaluated for their ability to determine gender in cell-free DNA from rhinoceros serum. Modifications to the original extraction and PCR protocols were required to obtain sufficient DNA quantities from serum, and both DNA yield and PCR amplification were substantially reduced or absent following multiple freeze-thaw cycles of serum. When fresh serum from 14 pregnant rhinoceroses (ultimately bearing seven male and seven female calves), representing four species at different stages of gestation (Days 61-490), were probed in a PCR-based assay, an accuracy of 71% was achieved for male-specific gene detection of SRY, which improved to 100% by including a reamplification step into the protocol. Such early sex determination should be a valuable tool for current management practices as well as future assisted reproduction of rhinoceroses.

Plant Genomic DNA Isolation-the Past and the Present, a Review

Successful isolation of a good quality of plant genomic DNA in sufficient quantity is a necessary prerequisite for its downstream applications, that are aimed to examine and analyze the molecular intricacies of the plant kingdom. The quantity and quality of isolated genomic DNA often determines the accuracy and robustness of results derived from many DNA based applications. The choice of the method adopted for DNA isolation depends on a large number of factors like the amount, nature and age of the starting plant tissue, the infrastructure available in terms of laboratory facilities, ready availability of required laboratory chemicals, time at hand and the downstream applications of the obtained DNA. A large number of modifications have been suggested to optimize DNA isolation from a particular plant species or tissue. There cannot be a universal protocol for DNA isolation across all plant species or tissues owing to the highly heterogeneous nature of plant cells. Hence, efforts need to be made to carry out suitable variations in the extraction method, so as to develop a protocol which is best suited to a particular plant species or tissue or application. So far, a large number of DNA isolation protocols from plants have been published, but there is no comprehensive report on suitability of these methods over each other and the different modifications which have been attempted to optimize the yield and quality of DNA. Many review papers have described various DNA isolation protocols and their underlying principles, that have been adopted in order to optimize DNA isolation from different plant species and tissues (Varma et al., 2007; Tan and Yiap 2009; Kumari et al., 2012). However, there is no comprehensive study which gives details of modifications at different steps of the plant genomic DNA isolation protocol, that have been attempted by researchers to overcome the specie and tissue specific limitations. This review gives a detailed account of the steps involved in genomic DNA isolation from various plant species and tissues and how each of these steps have been modified to overcome and eliminate the problem of contaminants in the extracted DNA so as to obtain a good quality of DNA amenable to downstream molecular biology applications.

Method for Detecting and Studying Genome-Wide Mutations in Single Living Cells in Real Time.

DNA sequencing and fluctuation test have been choice methods for studying DNA mutations for decades. Although invaluable tools allowing many important discoveries on mutations, they are both highly influenced by fitness effects of mutations, and therefore suffer several limits. Fluctuation test is for example limited to mutations that produce an identifiable phenotype, which is the minority of all generated mutations. Genome-wide extrapolations using this method are therefore difficult. DNA sequencing detects almost all DNA mutations in population of cells. However, the obtained population mutation spectrum is biased because of the fitness effects of newly generated mutations. For example, mutations that affect fitness strongly and negatively are underrepresented, while those with a strong positive effect are overrepresented. Single-cell genome sequencing can solve this problem. However, sufficient amount of DNA for this approach is obtained by massive whole-genome amplification, which produces many artifacts.We describe the first direct method for monitoring genome-wide mutations in living cells independently of their effect on fitness. This method is based on the following three facts. First, DNA replication errors are the major source of DNA mutations. Second, these errors are the target for an evolutionarily conserved mismatch repair (MMR) system, which repairs the vast majority of replication errors. Third, we recently showed that the fluorescently labeled MMR protein MutL forms fluorescent foci on unrepaired replication errors. If not repaired, the new round of DNA synthesis fixes these errors in the genome permanently, i.e., they become mutations. Therefore, visualizing foci of the fluorescently tagged MutL in individual living cells allows detecting mutations as they appear, before the expression of the phenotype.

Forensic DNA typing from teeth using demineralized root tips

Teeth are widely used samples in forensic human genetic identification due to their persistence and practical sampling and processing. Their processing, however, has changed very little in the last 20 years, usually including powdering or pulverization of the tooth. The objective of this study was to present demineralized root tips as DNA sources while, at the same time, not involving powdering the samples or expensive equipment for teeth processing. One to five teeth from each of 20 unidentified human bodies recovered from midwest Brazil were analyzed. Whole teeth were demineralized in EDTA solution with daily solution change. After a maximum of approximately seven days, the final millimeters of the root tip was excised. This portion of the sample was used for DNA extraction through a conventional organic protocol. DNA quantification and STR amplification were performed using commercial kits followed by capillary electrophoresis on 3130 or 3500 genetic analyzers. For 60% of the unidentified bodies (12 of 20), a full genetic profile was obtained from the extraction of the first root tip. By the end of the analyses, full genetic profiles were obtained for 85% of the individuals studied, of which 80% were positively identified. This alternative low-tech approach for postmortem teeth processing is capable of extracting DNA in sufficient quantity and quality for forensic casework, showing that root tips are viable nuclear DNA sources even after demineralization.

Forensic Dentistry – the key to the truth

Summary Introduction Human organism can be identified through testing and analysis of DNA sequences. The most common source of DNA for analysis is blood, soft tissues, hair, bones and teeth. Teeth represent a tissue of choice for analysis in those cases where there is high degree of degradation of other tissues. Hard tooth structure provides protection and preservation of DNA molecules. The aim was to investigate which group of teeth and dental tissue (pulp or hard dental tissues) has the greatest amount of DNA. Material and method Forty-five extracted teeth were analyzed. In the first examination 30 teeth were divided into the three groups (10 teeth each): first group were incisors, second premolars and third molars. The teeth were measured before and after the procedure of DNA isolation using special scale with precision of 0.02-0.000005ng. The procedure included grinding teeth in a blender and DNA isolation using commercial kits (isolation with magnetic particles). For the second test 15 teeth divided into two groups were used. In the first group isolation of DNA molecules was performed from pulp tissue, and in the second group from hard dental tissues. The quantification of samples was done with Quantifiler® Duo DNA Quantification Kit by Applied Biosystems. Results The greatest amount of DNA was obtained from molars (0.230011ng/μl/g) while the smallest amount of DNA was obtained from incisors and it was 0.06437ng/μl/g. In addition, the amount of DNA isolated from pulp tissue was significantly greater than that from hard dental tissues (pulp of molars obtained quantitatively the largest amount of DNA). Conclusion Main tissue to be used for the isolation of DNA from a tooth is pulp, but in those cases when it is not present (endodontic treatment), hard dental tissues provide sufficient quantity of DNA for identification procedures. The group of teeth that provides the greatest amount of DNA is molars.

Genetska identifikacija pogrešanih oseb

This article presents identification of missing persons from badly preserved post-mortem remains using molecular genetics methods. Extremely polymorphic and individually specific genetic markers that enable the identification of missing persons are microsatellites on autosomal chromosomes, microsatellites on Y chromosome and control region of mitochondrial DNA. For genetic profile comparison, biological material from post-mortem remains and reference samples have to be collected. If post-mortem remains are found shortly after the presumed death of the missing person, their personal items are used for comparison. If these are not available, (the missing person‘s) relatives could be used as reference samples or achieved tissues stored in medical institutions if biopsy for the needs of medical diagnostics was performed earlier during their life. When reference samples are not available, genetic identification is not possible. The type of biological material sampled from the deceased depends on the condition of human remains. Blood, soft tissues, nails, teeth or bones are most commonly used for genetic identification, and the time required for DNA extraction depends on the type of biological material. The most demanding and time consuming is extraction of DNA from teeth and bones, therefore we use it in cases when only skeleton is available or we cannot get a sufficient amount of DNA for genetic identification from other tissues. If the genetic profile of post-mortem reamains and a reference sample of the missing person match, the strength of genetic evidence has to be statistically evaluated and the probability of identification reported.

Formalin-fixed paraffin-embedded sample conditions for deep next generation sequencing

IntroductionPrecision medicine is only possible in oncology practice if targetable genes in fragmented DNA, such as DNA from formalin-fixed paraffin-embedded (FFPE) samples, can be sequenced using next generation sequencing (NGS). The aim of this study was to examine the quality and quantity of DNA from FFPE cancerous tissue samples from surgically resected and biopsy specimens. MethodsDNA was extracted from unstained FFPE tissue sections prepared from surgically resected specimens of breast, colorectal and gastric cancer, and biopsy specimens of breast cancer. A total quantity of DNA ≥60 ng from a sample was considered adequate for NGS. The DNA quality was assessed by Q-ratios, with a Q-ratio >0.1 considered sufficient for NGS. ResultsThe Q-ratio for DNA from FFPE tissue processed with neutral-buffered formalin was significantly better than that processed with unbuffered formalin. All Q-ratios for DNA from breast, colorectal and gastric cancer samples indicated DNA levels sufficient for NGS. DNA extracted from gastric cancer FFPE samples prepared within the last 7 years is suitable for NGS analysis, whereas those older than 7 years may not be suitable. Our data suggested that adequate amounts of DNA can be extracted from FFPE samples, not only of surgically resected tissue but also of biopsy specimens. ConclusionsThe type of formalin used for fixation and the time since FFPE sample preparation affect DNA quality. Sufficient amounts of DNA can be extracted from FFPE samples of both surgically resected and biopsy tissue, thus expanding the potential diagnostic uses of NGS in a clinical setting.

Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing

Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemiological studies. The aim of the present study was to standardize and compare two methods for the extraction of genomic DNA from sand flies for obtaining DNA in sufficient quantities for next-generation sequencing. Sand flies were collected from the municipalities of Campo Grande, Camapuã, Corumbá and Miranda, state of Mato Grosso do Sul, Brazil. Three protocols using a silica column-based commercial kit (ReliaPrep™ Blood gDNA Miniprep System kit, Promega®), and three protocols based on the classical phenol-chloroform extraction method (Uliana et al., 1991), were compared with respect to the yield and quality of the extracted DNA. DNA was quantified using a Qubit 2.0 fluorometer. The presence of sand fly DNA was confirmed by PCR amplification of the IVS6 region (constitutive gene), followed by electrophoresis on a 1.5% agarose gel. A total of 144 male specimens were analyzed, 72 per method. Significant differences were observed between the two methods tested. Protocols 2 and 3 of phenol-chloroform extraction presented significantly better performance than all commercial kit extraction protocols tested. For phenol-chloroform extraction, protocol 3 presented significantly better performance than protocols 1 and 2. The IVS6 region was detected in 70 of 72 (97.22%) samples extracted with phenol, including all samples for protocols 2 and 3. This is the first study on the standardization of methods for the extraction of DNA from sand flies for application to next-generation sequencing, which is a promising tool for entomological and molecular studies of sand flies.

A Multiplex PCR Assay for Differentiating Coconut Rhinoceros Beetle (Coleoptera: Scarabaeidae) From Oriental Flower Beetle (Coleoptera: Scarabaeidae) in Early Life Stages and Excrement.

The coconut rhinoceros beetle, Oryctes rhinoceros (L.), is a major pest of coconut and other palm trees. An incipient coconut rhinoceros beetle population was recently discovered on the island of Oahu, Hawaii and is currently the target of a large, mutiagency eradication program. Confounding this program is the widespread presence of another scarab beetle on Oahu, the oriental flower beetle, Protaetia orientalis (Gory and Percheron 1833). Eggs, early life stages, and fecal excrement of coconut rhinoceros beetle and oriental flower beetle are morphologically indistinguishable, thereby creating uncertainty when such specimens are discovered in the field. Here, we report the development of a multiplex PCR assay targeting cytochrome oxidase I of coconut rhinoceros beetle and oriental flower beetle that can rapidly detect and distinguish between these insects. This assay also features an internal positive control to ensure DNA of sufficient quantity and quality is used in the assay, increasing its reliability and reducing the chances of false negative results.

Library Construction from Subnanogram DNA for Pelagic Sea Water and Deep-Sea Sediments.

Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA.

Amelogenin Gene - The Pioneer in Gender Determination from Forensic Dental Samples.

In the event of any mass fatality incident, DNA analysis plays a vital role in disaster victim identification. Teeth are one of the most resistant structures in the human body that resist decomposition hence making them prime choice for extracting DNA for identification of individuals. Polymerase Chain Reaction (PCR) analysis that target regions of Amelogenin gene have become the method of choice for sex determination of biological samples. Determining the sex of a given DNA sample from either dental pulp or dentin of tooth and help in identification of missing persons and disaster victims. In our study 50 teeth samples were studied and they were subjected to various environmental conditions along with freshly extracted teeth taken as control for duration of one month and three months. Pulpal tissue was retrieved from the teeth specimens by access opening of root canals and for incinerated samples, the specimens were crushed. From the DNA that was extracted from the dental pulp sample Amelogenin gene locus was used for sex determination by amplifying a segment of X-Y homologous gene locus through PCR analysis. ANOVA test and t-test proved to be statistically significant and 100% retrieval rate was observed in samples. Pulpal tissue along with degenerating odontoblastic processes yield sufficient amount of DNA for gender determination when subjected to various forensic conditions with maximum accuracy.

Comparison of Clinically Relevant Mutation Profiles Between Preoperative Biopsy and Corresponding Surgically Resected Specimens in Japanese Patients With Non–Small-cell Lung Cancer by Amplicon-based Massively Parallel Sequencing

BackgroundAmplicon-based massively parallel sequencing (MPS) is an effective platform for identifying clinically actionable mutations across many genes in limited amounts of tissue. Most lung cancers are diagnosed and staged using small tissue samples obtained by transbronchial biopsy (TBB). To determine whether the mutations in TBB specimens detected by amplicon-based MPS reflect those present in the tumors, we compared the mutational profiles of preoperative TBB specimens and corresponding surgically resected specimens. Patients and MethodsFresh-frozen primary tumor specimens from non–small-cell lung cancer patients (n = 46) obtained preoperatively by TBB and during surgical resection were analyzed. The concordance of mutations detected by amplicon-based MPS in the 2 sample types was investigated, and the allele frequency of the mutations common to both specimens from the same patient was determined. ResultsAn initial assessment of DNA quantity revealed that 46% of the TBB specimens (21 of 46) had less than the lower limit for amplicon-based MPS. These 21 TBB specimens were consequently omitted from the analysis. Of the 29 mutations detected in the TBB and/or surgically resected specimens from 25 patients, 23 were present in both samples, for a concordance rate of 79%. ConclusionAmplicon-based MPS with TBB specimens approximately reflects clinically relevant tumor mutation profiles. However, the rate of TBB specimens with sufficient DNA quantity for amplicon-based MPS was only around 50%. Therefore, surgically resected specimens have a valuable role in exploratory and comprehensive genomic profiling.

Abstract 3620: Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction

Abstract Early detection and diagnosis of cancer substantially increases the likelihood for successful treatment. Tools that aid in detecting and diagnosing cancer early, therefore, have the potential to greatly impact the clinical outcome for cancer patients. Next Generation Sequencing (NGS) has emerged as an important tool in this area. The technology is sensitive, fast and high throughput to allow sequencing of many samples at once. Unfortunately, many clinical samples go unanalyzed because they do not yield sufficient quantities of DNA to generate NGS libraries or the libraries generated require so many rounds of PCR amplification that they display extreme sequence bias. Bias not only hampers data analysis, but also increases costs by requiring excess sequencing to obtain sufficient coverage over all relevant genomic regions. To enable the increased use of NGS in the clinic and reduce the amount of sequence bias generated during library preparation, we have developed a PCR free library construction method that uses low quantities of DNA as input. As an initial test of the method, we generated PCR free libraries from 100ng, 50ng and 25ng of human genomic DNA. The libraries where pooled and sequenced on the Illumina NextSeq 500 instrument to approximately 10X coverage. All libraries, irrespective of input amount, showed minimal AT/GC bias and excellent coverage distributions, with most bases covered within 5X of the expected coverage depth. In addition, regions identified as difficult to sequence (Aird, D., et.al., 2011 and Ross, M. G., et.al., 2013) showed coverage at near expected levels for all libraries. This method can easily be adapted for use with extremely low DNA inputs by the introduction of a minimal number of PCR cycles. In fact, we have used this method to construct high quality NGS libraries with picogram quantities of DNA input. Standard library construction methods require DNA inputs of 2ug to 500ng when PCR amplification is omitted. This new method utilizes inputs as low as 25ng to generate high-quality PCR free libraries and picogram quantities when amplification is performed. We are currently investigating the possibility of reducing input levels further and exploring the limits of the method with low quality DNA samples. Interestingly, we have observed substantial sample loss during DNA shearing and reaction cleanup. Samples that do not require fragmentation, such as DNA isolated from plasma (cfDNA) and low quality FFPE DNA, may reduce the input requirements even further. Finally, this new method utilizes low sample and reagent volumes, possibly paving the way for its use in microfluidic devices. Citation Format: Lynne Apone, Pingfang Liu, Vaish Panchapakesa, Deyra Rodriguez, Karen Duggan, Krishnan Keerthana, Nicole Nichols, Yanxia Bei, Julie Menin, Brad Langhorst, Christine Sumner, Christine Chater, Joanna Bybee, Laurie Mazzola, Danielle Rivizzigno, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3620.

SU‐F‐J‐07: Evaluating the Adequacy of Biopsy Specimens for Genetic Signature Assessment by Measuring the Metabolic Activity in Specimens Obtained Under 18F‐FDG PET/CT Guidance

Purpose:Genetic profiling of biopsied tissue is the basis for personalized cancer therapy. However biopsied materials may not contain sufficient amounts of DNA needed for analysis. We propose a method to determine the adequacy of specimens for performing genetic profiling by quantifying metabolic activity.Methods:We measured the response of two radiation detectors to the activity contained in the minimum amount of tumor cells needed for genetic profiling in biopsy specimens obtained under 2‐deoxy‐2‐(18F)fluoro‐D‐glucose (18F‐FDG) PET/CT guidance. The expected tumor cell concentration in biopsy specimens was evaluated from the amount of DNA needed (∼100 µg) and the number of pathology sections typically used for the analysis. The average 18F‐FDG uptake per cell was measured by incubating KPC‐4662 pancreatic tumor cells and HT‐29 colorectal adenocarcinoma tumor cells in 18F‐FDG containing solution (activity concentrations between 0.0122 and 1.51 MBq/mL and glucose concentrations of 3.1 and 1 g/L) for 1 to 1.75 hours and then measuring the activity of a known number of cells. Measurements of surrogate specimens obtained using 18G needle biopsies of gels containing these cells in expected concentrations (∼104 µL−1) were performed using an autoradiography CCD based device (up to 20 min exposure) and a scintillation well counter (∼1 min measurements) about 3 and 5 hours after the end of incubation respectively.Results:At start of autoradiography there were between 0.16 and 1.5 18F‐FDG molecules/cell and between 1.14 and 5.43×107 18F‐FDG molecules/mL. For the scintillation well counter, sample to minimum‐detectable‐count rate ratios were greater than 7 and the counting error was less than 25% for ≤80 s measurement times. Images of the samples were identifiable on the autoradiograph for ∼10 min and longer exposure times.Conclusion:Scintillation well counter measurements and CCD based autoradiography have adequate sensitivity to detect the tumor burden needed for genetic profiling in 18G core needle biopsies.Supported in part through the NIH/NCI Cancer Center Support Grant P30 CA008748 and by a sponsored research agreement with Biospace Lab S.A.

A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions.

Blood, saliva, and semen are some of the forensically most relevant biological stains commonly found at crime scenes, which can often be of small size or challenging due to advanced decay. In this context, it is of great importance to possess reliable knowledge about the effects of degradation under different environmental conditions and to use appropriate methods for retrieving maximal information from limited sample amount. In the last decade, RNA analysis has been demonstrated to be a reliable approach identifying the cell or tissue type of an evidentiary body fluid trace. Hence, messenger RNA (mRNA) profiling is going to be implemented into forensic casework to supplement the routinely performed short tandem repeat (STR) analysis, and therefore, the ability to co-isolate RNA and DNA from the same sample is a prerequisite. The objective of this work was to monitor and compare the degradation process of both nucleic acids for human blood, saliva, and semen stains at three different concentrations, exposed to dry and humid conditions during a 17-month time period. This study also addressed the question whether there are relevant differences in the efficiency of automated, magnetic bead-based single DNA or RNA extraction methods compared to a manually performed co-extraction method using silica columns. Our data show that mRNA, especially from blood and semen, can be recovered over the entire time period surveyed without compromising the success of DNA profiling; mRNA analysis indicates to be a robust and reliable technique to identify the biological source of aged stain material. The co-extraction method appears to provide mRNA and DNA of sufficient quantity and quality for all different forensic investigation procedures. Humidity and accompanied mold formation are detrimental to both nucleic acids.

The effect of mark enhancement techniques on the subsequent detection of saliva

There appears to be a limited but growing body of research on the sequential analysis/treatment of multiple types of evidence. The development of an integrated forensic approach is necessary to maximise evidence recovery and to ensure that a particular treatment is not detrimental to other types of evidence. This study aims to assess the effect of latent and blood mark enhancement techniques (e.g. fluorescence, ninhydrin, acid violet 17, black iron-oxide powder suspension) on the subsequent detection of saliva.Saliva detection was performed by means of a presumptive test (Phadebas®) in addition to analysis by a rapid stain identification (RSID) kit test and confirmatory DNA testing. Additional variables included a saliva depletion series and a number of different substrates with varying porosities as well as different ageing periods. Examination and photography under white light and fluorescence was carried out prior to and after chemical enhancement.All enhancement techniques (except Bluestar® Forensic Magnum luminol) employed in this study resulted in an improved visualisation of the saliva stains, although the inherent fluorescence of saliva was sometimes blocked after chemical treatment. The use of protein stains was, in general, detrimental to the detection of saliva. Positive results were less pronounced after the use of black iron-oxide powder suspension, cyanoacrylate fuming followed by BY40 and ninhydrin when compared to the respective positive controls. The application of Bluestar® Forensic Magnum luminol and black magnetic powder proved to be the least detrimental, with no significant difference between the test results and the positive controls. The use of non-destructive fluorescence examination provided good visualisation; however, only the first few marks in the depletion were observed. Of the samples selected for DNA analysis only depletion 1 samples contained sufficient DNA quantity for further processing using standard methodology. The 28-day delay between sample deposition and collection resulted in a 5-fold reduction in the amount of useable DNA. When sufficient DNA quantities were recovered, enhancement techniques did not have a detrimental effect on the ability to generate DNA profiles.This study aims to contribute to a strategy for maximising evidence recovery and efficiency for the detection of latent marks and saliva. The results demonstrate that most of the enhancement techniques employed in this study were not detrimental to the subsequent detection of saliva by means of presumptive, confirmative and DNA tests.

DNA transfer during laundering may yield complete genetic profiles

In a number of child sexual abuse cases, the alleged perpetrator is a member of the nuclear family. In those cases, there is a possibility that the suspect’s DNA was innocently deposited onto the child’s clothing without acts of sexual assault ever occurring, for example via secondary transfer within the washing machine. To assess the quantity and quality of DNA that may be transferred among clothing during laundering, we conducted three series of experiments. First, we evaluated the level of spermatozoa that may be transferred by washing pristine pairs of underwear with bed sheets containing a varying number of ejaculates. Secondly, we explored whether current genetic methods may also detect the transfer of DNA from vaginal secretions during a machine wash. Finally, we analyzed the background levels of DNA on children’s underwear collected from control families where sexual abuse never occurred. For both spermatozoa and vaginal secretions, we revealed that sufficient amounts of DNA may transfer onto laundered clothing to yield complete genetic profiles. Furthermore, DNA from relatives living within the same household was found in most cuttings taken from control children’s underwear. Based on these findings, we present a framework for the handling and interpretation of intrafamilial sexual abuse cases. These suggestions should help determine whether DNA was deposited directly onto a fabric or merely transferred during a wash.

Dielectrophoretic recovery of DNA from plasma for the identification of chronic lymphocytic leukemia point mutations.

Circulating cell free (ccf) DNA contains information about mutations affecting chronic lymphocytic leukemia (CLL). The complexity of isolating DNA from plasma inhibits the development of point-of-care diagnostics. Here, we introduce an electrokinetic method that enables rapid recovery of DNA from plasma. ccf-DNA was isolated from 25 µl of CLL plasma using dielectrophoresis. The DNA was used for PCR amplification, sequencing and analysis. The ccf-DNA collected from plasma of 5 CLL patients revealed identical mutations to those previously identified by extracting DNA from CLL cells from the same patients. Rapid dielectrophoresis isolation of ccf-DNA directly from plasma provides sufficient amounts of DNA to use for identification of point mutations in genes associated with CLL progression.

Decontamination by Persteril 36 may affect the reliability of DNA-based detection of biological warfare agents-short communication.

Persteril 36 is a disinfectant with a broad spectrum of antimicrobial activity. Because of its bactericidal, virucidal, fungicidal, and sporicidal effectiveness, it is used as a disinfectant against biological warfare agents in the emergency and army services. In case of an attack with potentially harmful biological agents, a person's gear or afflicted skin is sprayed with a diluted solution of Persteril 36 as a precaution. Subsequently, the remains of the biological agents are analyzed. However, the question remains concerning whether DNA can be successfully analyzed from Persteril 36-treated dead bacterial cells. Spore-forming Bacillus subtilis and Gram-negative Pseudomonas aeruginosa and Xanthomonas campestris were splattered on a camouflage suit and treated with 2 or 0.2% Persteril 36. After the disinfectant vaporized, the bacterial DNA was extracted and quantified by real-time PCR. A sufficient amount of DNA was recovered for downstream analysis only in the case of spore-forming B. subtilis treated with a 0.2% solution of Persteril 36. The bacterial DNA was almost completely destroyed in Gram-negative bacteria or after treatment with the more concentrated solution in B. subtilis. This phenomenon can lead to false-negative results during the identification of harmful microorganisms.