Impact of storage conditions and time on DNA yield from ammunition cartridges.

Recovery of suitable amounts of DNA from ammunition cartridges for short tandem repeat (STR) or mitochondrial (mt) DNA analysis has been a challenge for crime laboratories. The metal composition of cartridge cases and projectiles exposes the DNA to harmful ions that damage and ultimately degrade the DNA such that it cannot be effectively amplified. The current study assessed the impact of time and storage conditions on touch DNA deposited on cartridge components of varying metal content: aluminum, nickel, brass, and copper. Elevated humidity levels facilitated greater DNA degradation and loss compared to low humidity (or "dry") conditions, indicating that recovered cartridge component evidence should be stored in a low-humidity environment immediately after collection, preferably with a desiccant. As expected, a relationship was observed between the amount of time elapsed since the cartridge components were handled and the associated DNA yield. Interestingly, while yields dropped considerably in the first 48-96 h post-handling, regardless of the storage conditions, a layering effect was observed that helps maintain a relatively constant level of surface DNA over extended periods of time. An apparent layering effect was also observed on cartridge components following multiple surface depositions, where yields were two times higher than single deposition samples at similar timepoints. Overall, these findings suggest that storage conditions and a layering affect play an important role in the preservation of DNA on ammunition components.

Comparison of the performance of different models for the interpretation of low level mixed DNA profiles.

DNA analyses from forensic casework samples commonly result in complex DNA profiles. Often, these profiles consist of multiple contributors and display multiple stochastic events such as peak height imbalance, allelic or locus drop-out, allelic drop-in, and excessive or indistinguishable stutter. This increased complexity has established a need for more sophisticated methods of DNA mixture interpretation. This study compares the effectiveness of statistical models in the interpretation of artificially created low template two person mixed DNA profiles at varying proportions and template quantities. Two binary models (combined probability of inclusion and random match probability), a semicontinuous (Lab retriever), and continuous model (STRmix™) were compared. Generally, as the sophistication of the models increases, the power of discrimination increases. Differences in discrimination often correlate to each model's ability to use observed data effectively. Binary models require static thresholds resulting in unused data and outliers that may lead to difficult or incorrect interpretation. Semicontinuous and continuous models eliminate the stochastic threshold, however Lab Retriever does not account for stochastic events beyond drop-out and drop-in leading to possible less effective use of the data. STRmix™ incorporates all stochastic events listed above into the calculation making the most effective use of the observed data.

Study of CTS DNA Proficiency Tests with Regard to DNA Mixture Interpretation: A NIST Scientific Foundation Review.

The National Institute of Standards and Technology has released a document entitled DNA Mixture Interpretation: A NIST Scientific Foundation Review for public comment. This has become known as the Draft NIST Foundation Review. It contains the statement: "Across these 69 data sets, there were 80 false negatives and 18 false positives reported from 110,408 possible responses (27,602 participants × two evidence items × two reference items). In the past five years, the number of participants using PGS has grown." We examine a set of proficiency test results to determine if these NIST statements could be justified. The summary reports for each relevant forensic biology test (Forensic Biology, Semen, and Mixture) in the years 2018-2021 were reviewed. Data were also provided to us by CTS upon our request. None of the false positives or negatives could be attributed to the mixture interpretation strategy and certainly not to the use of PGS.

An improved process for the collection and DNA analysis of fired cartridge cases.

Improvements to the DNA analysis of fired cartridge cases have been made in recent years, yet successful analysis of this important evidence type remains difficult. In this study, we describe both a novel device for the collection and transport of fired cartridge cases and a new DNA recovery method that incorporates a rinse-and-swab technique. This technique combines two different types of swabs and a rinse solution with additives that reduce the degradative effects that copper has on DNA. The new recovery method yielded approximately threefold more DNA than the traditional double swab method and reduced the evidence of degradation. After validation, we estimated the real-world success rate of obtaining DNA profiles suitable for comparison with the rinse-and-swab method by testing over 100 cartridge cases collected from crime scenes. Approximately 67 % of the time (8 of 12), at least one DNA profile suitable for comparison was obtained from fired cartridge cases assumed to be associated with a single firearm using the collection device and the rinse-and-swab method when the fired cartridge cases were collected within 24 h.

Interpreting a major component from a mixed DNA profile with an unknown number of minor contributors.

Modern interpretation strategies typically require an assignment of the number of contributors (N) to a DNA profile. This can prove to be a difficult task, particularly when dealing with higher order mixtures or mixtures where one or more contributors have donated low amounts of DNA. Differences in the assigned N at interpretation can lead to differences in the likelihood ration (LR). If the number of contributors cannot reasonably be assigned, then an interpretation of the profile may not be able to be progressed. In this study, we investigate mixed DNA profiles of varying complexity and interpret them altering the assigned N. We assign LRs for true- and non- contributors and compare the results given different assignments of N over a range of mixture proportions. When a component of a mixture had a proportion of at least 10%, a ratio of at least 1.5:1 to the next highest component, and a DNA amount (as determined by STRmix™) of at least 50 rfu, the LR of the component for a true contributor was not significantly affected by varying N and was therefore suitable for interpretation and the assignment of an LR. LRs produced for minor contributors were found to vary significantly as the assigned N was changed. These heuristics may be used to identify profiles suitable for interpretation.

Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol's incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.

Application of random match probability calculations to mixed STR profiles.

Mixed DNA profiles are being encountered more frequently as laboratories analyze increasing amounts of touch evidence. If it is determined that an individual could be a possible contributor to the mixture, it is necessary to perform a statistical analysis to allow an assignment of weight to the evidence. Currently, the combined probability of inclusion (CPI) and the likelihood ratio (LR) are the most commonly used methods to perform the statistical analysis. A third method, random match probability (RMP), is available. This article compares the advantages and disadvantages of the CPI and LR methods to the RMP method. We demonstrate that although the LR method is still considered the most powerful of the binary methods, the RMP and LR methods make similar use of the observed data such as peak height, assumed number of contributors, and known contributors where the CPI calculation tends to waste information and be less informative.

An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA.

Damage to the DNA molecule can occur through exposure to environmental conditions such as ultraviolet light, heat and humidity. Forensic samples are particularly prone to such damage due to their prolonged exposure after deposition at crime scenes or mass disasters. Current methods for typing such samples rely heavily on the intact DNA template, and can be adversely affected by damage that is present. Proposed solutions center around increased access to the smaller remaining fragments and/or increased sensitivity. However, all rely on the polymerase chain reaction to copy the starting material; the required polymerase can be impeded by certain types of damage such as dimer-formation after ultraviolet light exposure, resulting in stochastic effects that can complicate interpretation. In vitro repair of such damage offers the ability to generate high quality profiles using traditional methods without changes to the current amplification reagents or conditions. Typically, repair reactions required large quantities of starting material and a separate repair reaction. Forensic samples, however, usually consist of small quantities, and quality control measures necessitate laboratory procedures that minimize sample manipulation. Here, an optimized protocol for forensic application of the PreCR™ Repair Mix to current typing methods is demonstrated for samples damaged by ultraviolet light exposure.

Effects of cyanoacrylate fuming, time after recovery, and location of biological material on the recovery and analysis of DNA from post-blast pipe bomb fragments*.

This study investigated the effects of time, cyanoacrylate fuming, and location of the biological material on DNA analysis of post-blast pipe bomb fragments. Multiple aliquots of a cell suspension (prepared by soaking buccal swabs in water) were deposited on components of the devices prior to assembly. The pipe bombs were then deflagrated and the fragments recovered. Fragments from half of the devices were cyanoacrylate fumed. The cell spots on the fragments were swabbed and polymerase chain reaction/short tandem repeat analysis was performed 1 week and 3 months after deflagration. A significant decrease in the amount of DNA recovered was observed between samples collected and analyzed within 1 week compared with the samples collected and analyzed 3 months after deflagration. Cyanoacrylate fuming did not have a measurable effect on the success of the DNA analysis at either time point. Greater quantities of DNA were recovered from the pipe nipples than the end caps. Undeflagrated controls showed that the majority (>95%) of the DNA deposited on the devices was not recovered at a week or 3 months.

Development of a quality, high throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks.

The attacks on the World Trade Center (WTC) Towers on September 11, 2001, represented the single largest terrorist-related mass fatality incident in the history of the United States. More than 2,700 individuals of varied racial and ethnic background lost their lives that day. Through the efforts of thousands of citizens, including recovery workers, medical examiners, and forensic scientists, the identification of approximately 1,500 victims had been accomplished through June 2003 (the majority of these identifications were made within the first 8-12 months). The principal role of The Bode Technology Group (Bode) in this process was to develop a quality, high throughput DNA extraction and short tandem repeat (STR) analysis procedure for skeletal elements, and to provide STR profiles to the Office of the Chief Medical Examiner (OCME) in New York City to be used for identification of the victims. A high throughput process was developed to include electronic accessioning of samples, so that the numbering system of the OCME was maintained; rapid preparation and sampling of skeletal fragments to allow for the processing of more than 250 fragments per day; use of a 96-well format for sample extraction, DNA quantification, and STR analysis; and use of the Applied Biosystems 3100 and 3700 instrumentation to develop STR profiles. Given the highly degraded nature of the skeletal remains received by Bode, an advanced DNA extraction procedure was developed to increase the quantity of DNA recovery and reduce the co-purification of polymerase chain reaction (PCR) amplification inhibitors. In addition, two new STR multiplexes were developed specifically for this project, which reduced the amplicon size of the STR loci, and therefore, enhanced the ability to obtain results from the most challenged of samples. In all, the procedures developed allowed for the analysis of more than 1,000 skeletal samples each week. Approximately 13,000 skeletal fragments were analyzed at least once, for a total of more than 18,000 analyses, and greater than 8,000 of the skeletal samples produced STR results (65%). The percentage of successful results was low in relation to previous mass fatality incidents involving airline disasters. However, when this same process was applied to the analysis of skeletal remains from the American Airlines Flight 587 disaster that occurred on November 12, 2001, the success rate was in line with expected results (ie, greater than 92% of the skeletal remains produced results). This illustrated the quality aspects of the procedure and the degree of degradation that had occurred for the remains of the WTC victims. For future mass fatality incidents, the quality, high throughput procedures developed by Bode will allow for more rapid DNA analysis of victim remains, more rapid identification of victims, and thus more rapid return of remains to family members.