Closed System DNA Purification for Degraded, Compromised Evidence in Microfluidic Devices

National Institute of Justice May 2013; Award Number: 2008-DN-BX-K144

Acquiring a DNA profile from large volume highly degraded or compromised forensic samples is particularly challenging sample type to be interfaced with a microfluidic system. Previous microfluidic devices utilizing a silica-based solid phase have been successful at purifying DNA from complex biological samples such as whole blood, however due to their small cross-sectional areas cannot operate at fast enough flow rates to make the processing of mL volumes a feasible option. The development of a volume reduction solid phase extraction (vrSPE) device employs a large cross-sectional area, enabling flow rates capable of handling a 0.5 mL sample in just ~30 minutes was realized. The large solid phase allows for greater binding sites on the silica surface, capable of binding the DNA but also the binding of PCR inhibitors, removing them from the eluate allowing for successful DNA profiling. In addition the vrSPE device reduces, and thus concentrates the DNA found in large volume samples often encountered throughout an investigation. Such samples include whole blood and semen stains, which need to be solubilized from the substrate prior to processing, diluting the DNA to concentrations in the order of 0.1 ng/μL. The vrSPE device was utilized to purify degraded whole blood and semen stains that were exposed up to 80°C for three months or subjected to UV light for the equivalent of 8 months and 16 days in LA sunlight, resulting in full STR profiles in each instance.

 

The extraction of bone was also demonstrated, a particularly difficult sample type due to the demoralization of the bone; a partial profile of 15 of 16 loci was produced. Once nuclear DNA is too degraded for STR amplification, mitochondrial DNA (mtDNA) can be used to identify persons. mtDNA is particularly susceptible to contamination, therefore an especially suitable sample type for the closed system microfluidic platform. The vrSPE device successfully purified and extracted amplifiable mtDNA from a severely heat degraded whole blood stain. The solid phase was also demonstrated to effectively remove inhibitors from the sample, specifically 35 μg of humic acid which is found in soil, outperforming current methods which see allelic dropout at 27 μg. The addition of a downstream μSPE phase, using chitosan-coated silica particles providing completely aqueous chemistry, further enhanced the removal of indigo dye – a common sample

contaminant found in blue jeans. STR profiles produced with the dual phase device were superior to those from the vrSPE channel alone, whereas a single μSPE channel failed to produce any peaks. This second phase offers two key advantages over one vrSPE alone – further removal of inhibitory material and increased concentration of the sample, especially resonant with severely diluted samples. Once the vrSPE method was fully established and demonstrated to be applicable to a plethora of sample types, fabrication of further devices in either a multi-channel or multiplex format were devised. For ease of fabrication and increased reproducibility between multiple channels, poly (methyl methacrylate) PMMA, was chosen as the new substrate for these devices. Following verification that PMMA operated as well as the previous glass substrate devices, a multiplex extraction of four separate dilute whole blood samples from four individuals was performed, resulting in full STR profiles. These results show the vrSPE technology is a successful method for processing a wide variety of large volume, degraded, inhibited biological samples in a timely, cross-contamination free manner.

Link to full-text report

If link does not work, request the report from Information & Research Services (QH Staff only)

Evaluating the Use of DNA and RNA Degradation for Estimating the Post-Mortem Interval

United States National Institute of Justice.  NIJ Grant Number 2010-DN-BX-K228

Estimating the post-mortem interval (PMI) is difficult due to the many factors that influence the decomposition process. Tissues such as nails, teeth and bones are more resilient to environmental factors. Measuring the rate of degradation of nucleic acids in these tissues could provide a method for estimating the PMI for longer time intervals, from days and weeks, through to months and years. These time intervals have not been studied before in a systematic manner using human tissues. Our initial focus has been on nails. Nails are hard tissues, relatively easy to sample and until now, have been under-utilized as a tissue that has forensic value. We have investigated the stability of nucleic acids in nails to determine if both DNA and RNA can be co-extracted in levels suitable for PCR analysis. Furthermore, multiple nail samples can be taken from the same individual so that measuring changes in nucleic acid degradation can be undertaken where there are a minimal number of external variables so that the nucleic acid degradation is a more likely representation of the PMI.

Using different methods for extracting DNA and RNA from nails, we have found that by modifying the Promega DNA IQ™ method, both DNA and RNA are efficiently extracted. This modified method has been implemented into operational forensic casework at ESR and nails are now being utilized in more human identification cases.

We have developed multiplex PCR assays to measure the rate of degradation of messenger RNA (mRNA), ribosomal RNA (rRNA) and DNA. Using nails placed in different environmental conditions (air, soil and water), we have found that nails are protected from other environmental factors and that nucleic acids (both DNA and RNA) can be amplified from samples left submerged in water or placed in soil for 120 days (1043 accumulated degree days). Nails from human cadavers have been collected, and the DNA and RNA co- extracted. Using our multiplex PCR assays, we have shown that DNA and RNA can be co-extracted from cadaver nails with a PMI up to 20,925 ADDs.

Using statistical analyses, some of the nucleic acid amplified fragments (from DNA and mRNA) look promising to use for deriving a statistical model for estimating the PMI.

Click to read full-text

If link does not work, request the report from Information & Research Services (QH Staff only)

Expected frequencies, exclusion percentages and ‘mathematical equivalence’: the probative value of DNA evidence in Aytugrul v The Queen

Australian Journal of Forensic Sciences Published online: 02 May 2013

In Aytugrul v The Queen the High Court held that it was acceptable to express the strength of DNA evidence in terms of an exclusion percentage. A key reason of the Court was that this was equivalent to the expected frequency of the DNA profile. This article assesses these two measures from a mathematical perspective. While the two are closely related, the frequency measure is to be preferred. The exclusion percentage fails to adequately capture the difference in probative value between DNA profiles of different frequencies.

Link to article

Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods

Science & Justice Available online 1 May 2013

Sex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples.

Click here to read full text

Enter the Library username and password at the login window (top right of screen). Contact the library if you require assistance.

Identification and Separation of Evidence Mixtures Using SNP-Based FISH Techniques and Laser Microdissection: Final Technical Report

United States National Institute of Justice A Bathrick et al; 30 July  2012;  NIJ Award #2009-DN-BX-K250

Laser microdissection (LM) has proven to be an effective method for cell mixture separations in the forensic laboratory. While sperm and epithelial cell sexual assault mixtures can be easily separated based upon morphological differences, mixtures of the same cell type are more difficult to separate. Past research has demonstrated that male/female cellular mixtures of similar morphology can be successfully separated using X/Y chromosome fluorescence in situ hybridization (FISH) probing. The goal of this research was to separate cellular mixtures of the same morphology and gender by developing FISH probes based on human genetic single nucleotide polymorphisms (SNPs). Screening panels of SNP FISH probes were intended to visually detect the individual contributors of sample mixtures, while laser microdissection would physically separate the cells for further STR processing. Padlock probes paired with tyramide signal amplification and rolling circle amplification were examined as methods of achieving this goal. Findings indicated that the FISH techniques utilized in this research were unsuitable for the detection of single SNP differences between individuals. Differentiation may be achieved in the future through research of other rolling circle amplification methods or by pursuing genetic marker systems that contain larger genetic differences. Multiple improvements in LM sample processing techniques were also examined. The use of cytogenetic on-slide lysis techniques and direct placement of LM collected cells into amplification reactions were evaluated as methods that would allow for the direct collection of nuclei and eliminate the need for DNA extraction prior to amplification. In addition, various amplification systems, amplification additives, and extraction techniques were investigated as alternative methods for processing samples in labs utilizing LM technologies. Although the on-slide lysis and direct amplification techniques were incompatible with this type of sample, the ZyGEM forensicGEM™ Saliva extraction and Promega’s PowerPlex® 16 HS System showed promise for work with LM collected samples. These techniques would also be ideal for labs attempting to process difficult evidence containing low copy number (LCN) cellular mixtures. The completed results obtained from this research will be disseminated through the law enforcement and scientific communities via seminars, journal articles, and poster presentations.

Link to full-text report

If link does not work, request the report from Information & Research Services (QH Staff only)

Identification and Separation of Same Gender Mixtures of Various Cell Types Using Interphase FISH Techniques and Laser Microdissection: Final Technical Report

United States National Institute of Justice A Bathrick et al; 29 May 2012;  NIJ Award #2008-IJ-CX-K016

The generation of clean, single source genetic profiles from sexual assault and touch evidence cellular mixtures is an enduring challenge in the field of forensics. Evidence of this nature can contain low copy amounts of DNA from mixtures of cell types of various morphologies. The goal of this research was to improve the methods of DNA mixture resolution in the forensic laboratory by improving laser microdissection (LM) techniques. Laser microdissection (LM) has proven to be an effective method for cell mixture separations in the forensic laboratory. While sperm and epithelial cell sexual assault mixtures can easily be separated based upon morphological differences, mixtures of the same cell type are more difficult to separate. The key objective of these studies was to separate cellular mixtures of similar morphology and same gender by using sequence specific fluorescence in situ hybridization (FISH) probes, which are based on the genetic polymorphisms associated with the Duffy and ABO blood groups. The Duffy and ABO blood grouping systems were chosen as the basis for these assays because of the existence of their prevalent polymorphisms throughout multiple populations. Each system has several alleles that can be targeted through a multicolor probe assay. The genetic sequences associated with the variations of each system are conserved and manageable in length. Thirty to three hundred base pair long FISH probes were designed to detect the single nucleotide polymorphisms observed between individuals of blood type A and blood type B. Tyramide signal amplification (TSA) was utilized to increase the sensitivity of FISH when detecting the short FISH probes. Before TSA-FISH techniques were employed, it was necessary to optimize individual steps for standard FISH detection as TSA cannot compensate for poor signals resulting from suboptimal pretreatment and hybridization conditions. For this research, epithelial cells from donors with blood groups A and B were used to examine the feasibility of TSA-FISH combined with LM as a method for separating cellular mixtures of similar morphology and same gender. Findings indicated that the FISH techniques utilized in this research were unsuitable for differentiation of the ABO blood groups; however, this could be achieved in the future pending research of other FISH methods or by pursuing other genetic marker systems that consist of larger genetic differences. The completed results will be disseminated through the law enforcement and scientific communities via seminars, journal articles, and poster presentations.

Link to full-text report

Untested evidence in sexual assault cases: using research to guide policy and practice

Sexual Assault Report Vol 16 no 3 Jan/Feb 2013 pp 33-48

We do not know how many unanalyzed SAKs there are, nationwide. There are many reasons for this, but one of the primary ones
i.s that tracking and counting SAKs is an antiquated process in many U.S. jurisdictions. A survey sponsored by the National Institute of Justice (NlJ) found that, as of 2007, 43% of the nation ‘s law enforcement agencies did not have a compmerized syslem for tmcking forensic evidence, either in their property room or after it was s.em to the crime lab.

Request the source article from Information & Research Services (QH Staff only)

Understanding how forensic science may contribute to miscarriages of justice

Australian Journal of Forensic Sciences 07 May 2013

Forensic professionals are now well accustomed to hearing from a variety of commentators about how they have contributed to wrongful convictions and miscarriages of justice.
The debate about the level and scale of the forensic ‘contribution’ continues with Simon Cole1 providing an in-depth review and comparison of two of the more recent commentators, Brandon Garrett2 and a counter perspective provided by John Collins and Jay Jarvis.

Link to full-text

Whose DNA is this? How relevant a question? (a note for forensic scientists)

Forensic Science International: Genetics Available online 25 April 2013

This communication seeks to draw the attention of researchers and practitioners dealing with forensic DNA profiling analyses to the following question: is a scientist’s report, offering support to a hypothesis according to which a particular individual is the source of DNA detected during the analysis of a stain, relevant from the point of view of a Court of Justice? This question relates to skeptical views previously voiced by commentators mainly in the judicial area, but is avoided by a large majority of forensic scientists. Notwithstanding, the pivotal role of this question has recently been evoked during the international conference “The hidden side of DNA profiles. Artifacts, errors and uncertain evidence” held in Rome (April 27th to 28th, 2012). Indeed, despite the fact that this conference brought together some of the world’s leading forensic DNA specialists, it appeared clearly that a huge gap still exists between questions lawyers are actually interested in, and the answers that scientists deliver to Courts in written reports or during oral testimony. Participants in the justice system, namely lawyers and jurors on the one hand and forensic geneticists on the other, unfortunately talk considerably different languages. It thus is fundamental to address this issue of communication about results of forensic DNA analyses, and open a dialogue with practicing non-scientists at large who need to make meaningful use of scientific results to approach and help solve judicial cases. This paper intends to emphasize the actuality of this topic and suggest beneficial ways ahead towards a more reasoned use of forensic DNA in criminal proceedings.

Click here to read full text

Enter the Library username and password at the login window (top right of screen). Contact the library if you require assistance.

 

Environmental DNA monitoring: beware of the transition to more sensitive typing methodologies

Australian Journal of Forensic Sciences Published online: 29 Apr 2013

Background DNA on surfaces and equipment utilised during forensic examination of exhibits poses a contamination risk. Many laboratories have established environmental DNA monitoring (EDM) programmes to generate awareness of locations, levels and origins of background DNA to identify contamination risk reduction opportunities. To evaluate the impact on contamination risk of introducing new DNA profiling systems we have analysed 134 EDM samples from surfaces within our forensic biology laboratory. The samples were profiled for our current system (Profiler Plus) and the new systems PowerPlex 21 and Yfiler. Many samples deemed clean using Profiler Plus yielded alleles using PowerPlex 21 and those with few alleles using Profiler Plus yielded many with PowerPlex 21. The detectability of alleles in multiple samples with negative quantification results using Quantifiler suggests that items able to be deemed free of detectable DNA by the newly introduced standard, AS 5483-2012, are not so when utilising DNA typing systems as sensitive as PowerPlex 21. Current cleaning procedures are shown to be less adequate when operating with PowerPlex 21. This study highlights the need for a range of issues pertaining to contamination risk to be addressed prior to the introduction of more sensitive profiling systems such as PowerPlex 21 into casework.

View the fulltext (QH staff only)