An enzymatic means for the rehabilitation of low-copy number and degraded DNA.

In spite of the immense success forensic DNA analysis has obtained over the last twenty-five years, a substantive challenge within the field of forensic DNA analysis is amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from minimal and poor quality biological evidence. It has been well established that DNA profiles obtained from degraded samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. Inspired by advances in next-generation sequencing techniques, I propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nucelase (DSN)

Analysis of heat-induced DNA damage during PCR and verification, validation and comparative analysis of two PCR megaplexes

Biological evidence collected at crime scenes are often subjected to forensic deoxyribonucleic acid (DNA) testing. During forensic DNA testing the DNA from the evidence and known samples are extracted, purified, amplified using Polymerase Chain Reaction (PCR), and analyzed using capillary electrophoresis (CE). In order to appropriately compare the profile of the suspect to the evidence, it is essential that interpretation parameters and optimized processing schemes are established. This study endeavors to accomplish this by: first, evaluating whether the PCR temperature cycling is detrimental to the amplification process; and second, by establishing and comparing interpretation parameters for two commonly employed short tandem repeat (STR) megaplexes. To evaluate the effects of temperature cycling on downstream signal, a dynamic systems model was developed, validated, and used to test the effects of temperature on DNA damage and the subsequent fluorescence signal. Though DNA is generally thought to be a stable molecule, heat-induced damage does occur. Specifically, this model assesses the damage to the guanine and cytosine bases during temperature cycling. The model conducts the amplification of a single locus during PCR and generates the peak height observed after capillary electrophoresis. The model was designed to assess not only the effects of heat-induced DNA damage but to also incorporate variability in PCR efficiency. The simulated data indicate that heat-induced DNA damage does not significantly reduce the allelic signal. Also, although changes in PCR efficiency introduce variability in the peak heights at all targets, the peak heights observed with and without heat-induced DNA damage are not significantly different. In fact, the variation in PCR efficiency has a larger effect on the number of amplicons produced than does the heat-induced DNA damage. The second part of this study compares two PCR amplification megaplexes, PowerPlex® Fusion and GlobalFiler®, by evaluating their sensitivities, limits of detection, presence of artifacts, heterozygous peak balance, and ability to amplify minor contributors in DNA mixtures. Analysis of single source samples using weighted least squares regression analysis indicates that PowerPlex® Fusion has greater analytical sensitivities and lower limits of detection at comparable dye channels, and both kits display similar heterozygous balance. However, the GlobalFiler® processing scheme produced fewer artifacts for the various single source samples analyzed, particularly at higher target amounts. Also, analysis of two and three person DNA mixtures indicates that both megaplexes perform equally well when detection of the minor contributor is the criterion

Examining peak height ratios in low template DNA samples with and without sampling using a single-tube extraction protocol

The developments of the polymerase chain reaction (PCR) and the short tandem repeat multiplex kits increased the ease and lowered the time and sample quantity required for deoxyribonucleic acid (DNA) typing compared to previous methods. However the amplification of low mass of DNA can lead to increased stochastic effects, such as allele drop-out (ADO) and heterozygous peak height (PH) imbalance, which make it difficult to determine the true donor profile. These stochastic effects are believed to be due to: 1) pre-PCR sampling from pipetting and sample transferal of dilute samples prior to amplification resulting in unbalanced heterozygous allele templates in the amplification reaction, and 2) the kinetics of the PCR process where, when few target templates are available, there is uneven amplification of heterozygous alleles during early PCR cycles. This study looks to examine the contribution of PCR chemistry and pre-PCR sampling errors on stochastic effects by utilizing a single-tube DNA extraction and direct amplification method. Cells were collected into tubes using the McCrone and Associates, Inc. cell transfer method, which allowed for approximation of DNA mass without quantification. The forensicGEM® Saliva Kit was used to lyse the cells and inactivate nucleases without inhibiting downstream amplification. The samples were then directly amplified with the AmpFLSTR® Identifiler® Plus PCR Amplification Kit. These samples should only show the effects of PCR chemistry since pipetting and tube transferal steps prior to amplification were removed with the expectation that equal numbers of heterozygous alleles are present in the sample pre-amplification. Comparisons of PH imbalance were made to samples extracted with forensicGEM® but had one or more pipetting and tube transferal steps prior to amplification. These samples were either created through the dilution of stock DNA or from the cell transfer method where aliquots were then taken for amplification; thus these samples would exhibit the effects of both pre-PCR sampling and PCR chemistry errors and inefficiencies. The use of carrier ribonucleic acid (cRNA) was also added to cell transfer samples prior to the amplification of samples to see if it assisted with amplification and increased signal. Results show that the samples with only PCR chemistry generally have significantly higher mean peak height ratios (PHRs) than samples with both pre-PCR sampling and PCR chemistry except in cases where there were large numbers of ADOs. When compared to the diluted samples, the cell transfer samples had significantly higher mean PHR at 0.0625 ng and 0.125 ng, and higher mean PHR at 0.0375 ng when PHs from ADOs are included. Average peak heights (APHs) in the cell transfer samples were also significantly higher in these comparisons. When compared to aliquots taken from cell transfer samples, mean PHR was significantly higher at 0.0625 ng in cell transfer samples with only PCR chemistry than cell transfer samples with both pre-PCR sampling and PCR chemistry; however APH for the samples with only PCR chemistry was also significantly higher in one experiment and not significantly different in another. In a third experiment, the difference in mean PHR was not significant while APH was significantly higher in the samples with pre-PCR sampling and PCR chemistry; however there were also a large numbers of ADOs. Our results also found quantification of dilute samples unreliable but cell counting through the cell transfer method is an appropriate alternative for DNA mass approximation. Also there were no significant changes in PHR or APH in the presence or absence of cRNA

Characterizing double-back stutter in low to multi-copy number regimes in forensically relevant STR loci

Modern DNA analysis is possible due to the discovery of repeating microsatellite regions in DNA and successful implementation of the polymerase chain reaction (PCR) in laboratories. PCR amplification chemistries that contain short tandem repeat (STR) loci are sensitive. As a result, the discrimination power within human identification sciences has increased in recent years. Despite these advances, cellular admixtures are commonly collected, and the resultant “DNA mixture profile” is difficult to interpret as it is often encumbered by low-signals and allele drop-out. Regularly detected PCR artifacts can further complicate interpretation. One commonly encountered artifact is stutter, the result of strand slippage during PCR. Stutter can be of two types: forward and reverse. Reverse stutter (or back stutter) is the most prevalent and is one repeat unit shorter (n - 1) than the template strand. In contrast, forward stutter is one repeat unit longer (n + 1). If a reverse stutter amplicon is produced there is the distinct possibility that a stutter product of stutter may occur. This artifact is usually referred to as double-back stutter (DBS) or n - 2 stutter. Recently there has been renewed interest in examining signal approaching baseline levels. As the sensitivity of the process improves, so does the probability of detecting DBS. Therefore, studies that examine the peak height distributions, rarity, stutter signal-to-noise distances and the general impact of DBS on the signal are warranted. Models simulating PCR, and the entire forensic DNA process, have been created by this laboratory. The work presented herein builds upon a preexisting model; specifically, the dynamic model was extended such that DNA profiles consisting of 21 autosomal STRs, consistent with the GlobalFilerTM multiplex, are simulated. Furthermore, this expansion incorporated a three-type Galton-Watson branching process allowing DBS to be added to the simulated electropherogram (EPG). The in silico model was used to simulate the amplification of a 1:43 and 1:73 mixture at a total DNA concentration of 0.3 and 0.5 ng, respectively. We chose these extreme mixture ratios because the signal from these minor contributors would be most susceptible to DBS effects from the major contributor. A total of 1200 alleles from each contributor were simulated at each target, and effects of DBS on the signal from the minor contributor were characterized. At 0.3 and 0.5 ng both the noise and stutter signal histograms are right-skewed and a Kolmogorov-Smirnov (KS) test indicates that the noise and DBS were significantly different (p-value < 4x10-6). The average peak height of DBS for all loci in both scenarios were less than 50 RFU (Relative Fluorescence Units), and the DBS ratios ranged from 0.29 to 2.15% of the main allele, with the median ratios less than 0.5%. A per locus analytical threshold (AT) was calculated for both the 0.3 and 0.5 ng targets using two k-values: 3 and 4. The k-value is chosen based on the Type I risk assessment, wherein increasing the k-value increases AT. The percentage of DBS peaks greater than AT when k = 3 for the mixtures amplified at 0.3 and 0.5 ng ranged from 0 to 7.08% and 0 to 10.50%, respectively. Interestingly, when k = 4 the percentage of DBS peaks greater than AT for 0.3 and 0.5 ng reduced to 0 to 1.08% and 0 to 0.17%, respectively. This suggests that modeling DBS in continuous systems may not be necessary if the laboratory continues to rely on a system that requires an AT of sufficient strength. However, with the advent of Bayesian or machine learning-based approaches to analyzing EPGs, thus removing AT in its entirety, a complete understanding of the prevalence of DBS is necessary. This work shows that DBS from an extreme major using our laboratory protocols is not likely to be in the same signal regime as the signal from alleles; however, it does show that signal from DBS is significantly different from noise. Therefore, the software expert pair should be carefully considered during the validation stage and laboratories should consider DBS during interpretation, especially if enhanced post-PCR parameters are implemented into the forensic laboratory process

Determining the change in PCR efficiency with cycle number and characterizing the effect of serial dilutions on the DNA signal

The ability to obtain deoxyribonucleic acid (DNA) profiles is generally considered a powerful tool when examining evidence associated with a crime scene. However, variability in peak heights associated with short tandem repeats (STR) signal complicates DNA interpretation; particularly, low-template complex mixtures, which are regularly encountered during evidentiary analysis. In order to elucidate the sources that cause peak height variability a dynamic model, which simulates; 1) the serial dilution process; 2) polymerase chain reaction (PCR); and 3) capillary electrophoresis (CE) was built and used to generate simulated DNA evidentiary profiles. In order to develop the dynamic model, PCR efficiencies were characterized. This was accomplished using empirical quantitative polymerase chain reaction (qPCR) data. Specifically, the ratios of fluorescent readings of two consecutive cycles were evaluated. It was observed that the efficiency fluctuated at early cycles; stabilized during the middle cycles; and plateaued during later cycles. The relationship between the change in efficiency and the concentration of amplicons was modeled as an exponential function. Subsequently, this exponential relationship was incorporated into the dynamic model as a part of the PCR module. Using the dynamic laboratory model, the effect of serially diluting a concentrated DNA extract to a low-template concentration was assessed in an effort to determine whether serially diluted samples are a good representation of evidence samples which contain low copy number of cells. To accomplish this, peak height variances and the frequency of drop-out between serially and non-serially diluted samples were compared. The results showed that diluting the sample had a substantial influence on allelic drop-out. However, the distributions of the observed peak heights did not consistently change; though, changes in peak height distributions became more pronounced with samples at lower targets. The peak height equivalency (PHE) was also used to aid in the determination of the effect of serial dilutions on reproducibility. There was not a major change in PHE between serially and non-serially diluted samples

Revolutionizing genomic analyses: mutation analyses using novel enzyme-based assays with laser-induced fluorescence and polymeric microfluidic devices as electrophoretic platforms

Polymer-based microelectrophoresis was investigated to analyze known (mutation detection) and unknown (mutation scanning) low-abundant mutations in genomic DNA with high diagnostic value for colorectal cancers. For our mutation detection assays, point mutations in the K-ras oncogene were identified using the ligase detection reaction (LDR). For the mutation scanning assay, which searches for sporadic mutations, an EndoV-LDR assay was utilized with mutations in the p53 tumor suppressor gene used as a model. A poly(methylmethacrylate), PMMA, microchip filled with a 4% linear polyacrylamide (LPA) gel was used to electrophoretically sort products formed from LDRs, which produced oligonucleotides \u3c65 bp in length. Using microchip electrophoresis with the LPA, a 44 bp ligation product was resolved from a 100-fold molar excess of unligated primers (25 bp) in approximately 120 s, which was ~17 times faster than conventional capillary gel electrophoresis. In order to simplify the electrophoretic process and further reduce development time, the LDR products were sorted in the absence of the sieving gel using free solution conjugate electrophoresis (FSCE). FSCE incorporated polyamide “drag-tags” onto LDR primers, which provided DNA fragment mobilities in free solution that were dependent upon their size. LDR/drag-tagged (LDR-dt) products could be formed in a multiplexed format for mutant-to-wild-type ratios as low as 1 to 100 with single base resolution. Separations were conducted using capillary array electrophoresis (CAE) and PMMA microchips filled with only a TRIS buffer. Analysis times for the LDR-dt products were less than 11 min using CAE and ~85 s for PMMA microchips with high reproducible migration times within and between microchips. PMMA-based microchips were also evaluated for the identification of sporadic mutations using an endonuclease V – LDR (Endo V/LDR) technique. Endo V cleaves heteroduplexed DNA one base 3’ of single-base mismatched sites as well as nicking DNA at some matched sites as LDR reseals miscleaved sites to reduce false positive signals. Results suggested that Endo V/LDR products from p53 mutations could be successfully separated and detected using a PMMA microfluidic chip filled with a sparsely cross-linked replaceable polyacrylamide gel in less than 6 min, which was approximately 10-fold shorter compared to CAE

Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization

Initiation of mineralization during endochondral ossification is a multistep process and has been assumed to correlate with specific interactions of annexins A5 and A6 and collagens. However, skeletal development appears to be normal in mice deficient for either A5 or A6, and the highly conserved structures led to the assumption that A5 and A6 may fulfill redundant functions. We have now generated mice deficient of both proteins. These mice were viable and fertile and showed no obvious abnormalities. Assessment of skeletal elements using histologic, ultrastructural, and peripheral quantitative computed tomographic methods revealed that mineralization and development of the skeleton were not significantly affected in mutant mice. Otherwise, global gene expression analysis showed subtle changes at the transcriptome level of genes involved in cell growth and intermediate metabolism. These results indicate that annexins A5 and A6 may not represent the essential annexins that promote mineralization in vivo

Screening Sexual Assault Evidence with Low Concentrations of Male DNA Utilizing the RapidHIT 200 and ParaDNA Intelligence Test

Over the last several years, crime laboratories have largely focused on the sexual assault kit (SAK) backlog, where they are often confronted with many low-quality samples. The lack of available screening techniques has prevented analysts from gaining insight into the disposition of the sample earlier on in the testing process; requiring analysts to rely on visual observations and little background information. Consequently, resulting in the hindrance of probative STR profiles while expending a large amount of time and resources to gain this result. In recent years, crime laboratories have explored a male screening technique, recommended by SWGDAM, to combat this problem. However, it is important to investigate alternative screening methods that can be performed prior to receiving the evidence at a crime laboratory, allocating time and resources toward enhanced testing of probative samples. Mock sexual assault admixtures were prepared at different mixture ratios and split into two data sets. The first data set was comprised of 24 total samples that were prepared at the 1:10 and 1:20 mixture ratio, then run on the RapidHIT® 200. The second data set consisted of 91 total samples that were prepared at the following mixture ratios: 1:5, 1:10, 1:15, 1:20, 1:25. These samples were then run on the ParaDNA® System with the Intelligence Test assay. The results of this study showed that the RapidHIT® 200 outperformed the ParaDNA® Intelligence Test when utilized to screen sexual assault admixtures in a non-laboratory setting. The RapidHIT® 200 was successful at detecting a mixture profile in 75% of the mock sexual assault cases. Whereas, the ParaDNA® Intelligence Test had less than one in four chance of detecting the presence of a mixture. In other words, the linear mixed-effects model demonstrated that the instrument used for screening sexual assault samples in a non-laboratory setting does have a significant effect on the proportion of loci that exhibit a mixture. In conclusion, the ParaDNA® Intelligence Test is not recommended for use in a non-laboratory setting. Although the RapidHIT® 200 outperformed this test, further research should be conducted before use in any setting

Characterizing low copy DNA signal using simulated and experimental data

Sir Alec Jeffreys was the first to describe human identification with deoxyribonucleic acid (DNA) in his seminal work in 1985 (1); the result was the birth of forensic DNA analysis. Since then, DNA has become the primary substance used to conduct human identification testing. Forensic DNA analysis has evolved since the work of Jeffreys and now incorporates the analysis of 15 to 24 STR (short tandem repeat) locations, or loci (2-4). The simultaneous amplification and subsequent electrophoresis of tens of STR polymorphisms results in analysis that are highly discriminating. DNA target masses of 0.5 to 2 nanograms (ng) are sufficient to obtain a full STR profile (4); however, pertinent information can still be obtained if low copy numbers of DNA are collected from the crime scene or evidentiary material (4-9). Despite the sensitivity of polymerase chain reaction (PCR) - capillary electrophoresis (CE) based technology, low copy DNA signal can be difficult to interpret due to the preponderance of low signal-to-noise ratios. Due to the complicated nature of low template signal, optimization of the DNA laboratory process such that high-fidelity signal is regularly produced is necessary; studies designed to effectively hone in on optimized laboratory conditions are presented herein. The STR regions of a set of samples containing 0.0078 ng of DNA were amplified for 29 cycles; the amplified fragments were separated using two types of CE platforms: an ABI 3130 Genetic Analyzer and an ABI 3500 Genetic Analyzer. The result is a genetic trace, or electropherogram (EPG), comprised of three signal components that include noise, artifact, and allele. The EPGs were analyzed using two peak detection software programs. In addition, a tool, termed Simulating Evidentiary Electropherograms (SEEIt) (10, 11), was utilized to simulate EPG signal obtained when one copy of DNA is processed through the forensic pipeline. SEEIt was parameterized to simulate data corresponding to two laboratory scenarios: the amplification of a single copy of DNA injected on an ABI 3130 Genetic Analyzer and on an ABI 3500 Genetic Analyzer. In total, 20,000 allele peaks and 20,000 noise peaks were generated for each CE platform. Comparison of simulated and experimental data was used to elucidate features that are difficult to ascertain by experimental work alone. The data demonstrate that experimental signal obtained with the ABI 3500 platform results in signal that is, on average, a factor of four larger than signal obtained from the ABI 3130 platform. When a histogram of the signal is plotted, a multi modal distribution is observed. The first mode is hypothesized to be the result of noise, while the second, third, etc. modes are the signal obtained when one, two, etc. target DNA molecules are amplified. By evaluating the data in this way, full signal resolution between noise and allelic signal is visualized. Therefore, this methodology may be used to: 1) optimize post-PCR laboratory conditions to obtain excellent resolution between noise and allelic signal; and 2) determine an analytical threshold (AT) that results in few false detections and few cases of allelic dropout. A χ2 test for independence of the experimental signal in noise positions and the experimental signal within allele positions < 12 relative fluorescence units (RFU), i.e. signal in the noise regime, indicate the populations are not independent when sufficient signal-to-noise resolution is obtained. Once sufficient resolution is achieved, optimized ATs may be acquired by evaluating and minimizing the false negative and false positive detection rates. Here, a false negative is defined as the non-detection of an allele and a false positive is defined as the detection of noise. An AT of 15 RFU was found to be the optimal AT for samples injected on the ABI 3130 for at least 10 seconds (sec) as 99.42% of noise peaks did not exceed this critical value while allelic dropout was kept to a minimum, 36.97%, at this AT. Similarily, in examining signal obtained from the ABI 3500, 99.41% and 99.0% of noise fell under an AT of 50 RFU for data analyzed with GeneMapper ID-X (GM) and OSIRIS (OS), respectively. Allelic dropout was 36.34% and 36.55% for GM and OS, respectively, at this AT

Improving DNA evidence collection via quantitative analysis: a systems approach

Thesis (M.A.)--Boston UniversityWhen collecting biological evidence from a crime scene, it is important to determine the most effective and robust collection method to ensure maximum DNA recovery. Some common biological collection methods include swabbing, cutting, scraping, and taping. Although these techniques have been a mainstay of forensic analysis, each of these methods have significant drawbacks, which include but are not limited to, the lack of surface area that may be processed, possible co-elution of PCR inhibitors, and non-optimized elution of cells from the substrate into solution. Therefore, a technique designed to optimize biological collection from items of interest, particularly large items, is necessary and not currently available for forensic use