11 research outputs found
Evaluation of nucleosome forming potentials (NFPs) of forensically important STRs
Degraded forensic samples have proved difficult to analyze and interpret. New analysis techniques are constantly being discovered and improved but researchers have overlooked the structural properties that could prevent or slow the process of degradation. In theory, DNA that are bound to histones as nucleosomes are less prone to degradation, because nucleosomes prevent DNA from being exposed to degradative enzymes. In this study we determined the probability of 60 forensic DNA markers to be bound to histones based on their base sequence composition. Two web-based tools - NXSensor and nuScore - were used to analyze four hundred base pairs surrounding each DNA marker for properties that inhibit or promote the binding of DNA to histones. Our results showed that the majority of markers analyzed were likely to be bound as nucleosomes. Selection of the markers that are more protected to form a multiplex could increase the chance of obtaining a better balanced, easier to interpret DNA profile from degraded sample
Heptaplex-direct PCR assay for simultaneous detection of foodborne pathogens
ÂĐ 2017 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/
This author accepted manuscript is made available following 12 month embargo from date of publication (Sept 2017) in accordance with the publisherâs archiving policyFoodborne pathogens pose significant problems for public health and economy. The gold standard, cultivation, is time-consuming and costly. In this study, a heptaplex-direct PCR assay for simultaneous detection of seven foodborne pathogens without DNA extraction and enrichment was developed and validated. Seven virulent genes of target strains were amplified and found that the assay provided the expected PCR fragment of 583, 490, 415, 343, 224, 209, and 105 bp for Shigella spp., Shiga toxin-producing Escherichia coli (STEC), Streptococcus pyogenes, Campylobacter jejuni, Salmonella Typhi, Listeria monocytogenes, and Staphylococcus aureus, respectively. Validation study showed that the assay was highly reproducible, specific and sensitive (106â100 CFU/ml of detection limit). Moreover, assay application on 22 artificially-contaminated and 100 food samples provided a statistically equivalent efficiency to the culture method. A heptaplex-direct PCR assay thus can be used in microbial forensic science
Degradation, quantification and the theory of nucleosome protection
Partial DNA profiles are often obtained from degraded samples due to allelic and locus dropout, particularly at the high molecular weight loci. Increasing the chance of genotyping success via shortening of amplicon size has been previously demonstrated with mini-STRs. A viable alternative based on nucleosome protection influenced by base sequences was explored in this study. Different systems for accurate quantification of degraded samples were looked at, including both single- and multi-copy targets. Optimisation was successful for GAPDH and it was compared to PlexorÂŪ HY using casework samples. Although GAPDH was more accurate, PlexorÂŪ HY was chosen and used to quantify degraded saliva samples due to its higher sensitivity and informativeness. The saliva samples had been degraded via the incubation method, which was assumed to preserve the chromatin structure. Next-generation and mini-STR kits were assessed in terms of sensitivity and casework genotyping. All kits performed exceptionally well and were comparable in all categories. 60 sequences (58 STRs and amelogenin X and Y) were evaluated for their nucleosome-forming potential (NFP) using two computer programs. The markers were divided into three groups based on their NFPs and the findings were verified empirically by amplifying degraded saliva samples and casework samples using 14 randomly chosen primer sets from the three groups. The effect of nucleosome protection was not observed for degraded saliva and casework samples. This is the first study that looks at an inherent property of STRs as a determinant of survivability from degradation processes. The work can be expanded to include more sample types. Other computer programs can be used, as predicting nucleosome positions is a rapidly advancing field.Partial DNA profiles are often obtained from degraded samples due to allelic and locus dropout, particularly at the high molecular weight loci. Increasing the chance of genotyping success via shortening of amplicon size has been previously demonstrated with mini-STRs. A viable alternative based on nucleosome protection influenced by base sequences was explored in this study. Different systems for accurate quantification of degraded samples were looked at, including both single- and multi-copy targets. Optimisation was successful for GAPDH and it was compared to PlexorÂŪ HY using casework samples. Although GAPDH was more accurate, PlexorÂŪ HY was chosen and used to quantify degraded saliva samples due to its higher sensitivity and informativeness. The saliva samples had been degraded via the incubation method, which was assumed to preserve the chromatin structure. Next-generation and mini-STR kits were assessed in terms of sensitivity and casework genotyping. All kits performed exceptionally well and were comparable in all categories. 60 sequences (58 STRs and amelogenin X and Y) were evaluated for their nucleosome-forming potential (NFP) using two computer programs. The markers were divided into three groups based on their NFPs and the findings were verified empirically by amplifying degraded saliva samples and casework samples using 14 randomly chosen primer sets from the three groups. The effect of nucleosome protection was not observed for degraded saliva and casework samples. This is the first study that looks at an inherent property of STRs as a determinant of survivability from degradation processes. The work can be expanded to include more sample types. Other computer programs can be used, as predicting nucleosome positions is a rapidly advancing field
Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I
Here, we applied the Taguchi method, an engineering optimization process, to successfully determine the optimal conditions for three SYBR Green I-based quantitative PCR assays. This method balanced the effects of all factors and their associated levels by using an orthogonal array rather than a factorial array. Instead of running 27 experiments with the conventional factorial method, the Taguchi method achieved the same optimal conditions using only nine experiments, saving valuable resources
āļĢāļēāļĒāļāļēāļāļ§āļīāļāļąāļĒāļāļāļąāļāļŠāļĄāļāļđāļĢāļāđāđāļāļĢāļāļāļēāļĢāļāļēāļĢāļāļĢāļ§āļāļāļĩāđāļāđāļāđāļāļāļēāļāļāļāļāļāļĢāļ°āļŠāļļāļ āļāļĢāļ°āļŠāļļāļ āđāļĨāļ°āļāļĨāļāļāļāļĢāļ°āļŠāļļāļ āļāđāļ§āļĒāļ§āļīāļāļĩāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđ
Cartridges, bullets, and casings (CBCs) are commonly encountered in shooting incidences and could provide valuable DNA information from touch DNA that has been left during bullet handling and gun loading; however, conventional DNA analysis has yielded very poor results. Direct PCR, in which the DNA extraction and quantification steps are bypassed, has been shown to provide comparable and sometimes improved results from touch DNA and trace DNA samples. Here, we aimed to apply direct PCR with bullet casings (from three ammunition types and guns) and evaluate whether it should be recommended as a standard operating protocol for forensic DNA analysts. Three experiments were carried out to investigate the following: the effect of firing on DNA deposited on bullet casings; the effect of gun and ammunition types on STR profile quality; and the feasibility of using direct PCR with actual cases via typing of mock casework samples. DNA extraction resulted in a loss of about 40% of DNA originally deposited, and firing a bullet decreased the amount of DNA recovered by 27%. We recovered means (and 95% credible intervals) of 11.1 (7.9 to 13.9), 5.6 (3.0 to 7.7), 2.3 (0.2 to 4.0) alleles from touch DNA on fired bullet casings using the direct PCR protocol, conventional extraction protocol, and dilution protocol, respectively. No statistical difference in alleles recovered was observed between different fired ammunition types from three guns (9mm, 7.62 mm, and 5.56 mm from Glock Model 19, AK47, and Tavor T-21, respectively). As expected, mixed DNA profiles were observed in 40% of mock casework samples in which guns are shared between volunteers, which can complicate profile interpretation. This study showed that direct PCR from bullet casings improved STR profiles. As the direct PCR protocol is quicker, cheaper, and resulted in more alleles recovered, forensic DNA analysts may benefit from using direct PCR.āļāļĢāļ°āļŠāļļāļāļāļ·āļāđāļĨāļ°āļāļĨāļāļāļāļĢāļ°āļŠāļļāļāļāļ·āļāļāļđāļāļāļāđāļāđāļāđāļāļĒāđāļāļāļĩāđāđāļāļīāļāđāļŦāļāļļāļāļāļāļāļāļĩāļāļ§āļēāļĄāļāļĩāđāļĄāļĩāļāļēāļĢāđāļāđāļāļ·āļ āļāļķāđāļāļāļēāļāļāļ°āđāļāđāļ āđāļŦāļĨāđāļāļāļāļāļāļĩāđāļāđāļāđāļāļāļĩāđāđāļāļīāļāļāļēāļāļāļēāļĢāļŠāļąāļĄāļāļąāļŠāļĢāļ°āļŦāļ§āđāļēāļāļāļēāļĢāļāļĢāļĢāļāļļāļāļĢāļ°āļŠāļļāļāļĨāļāļāļāļāļāļĢāļ°āļŠāļļāļāļŦāļĢāļ·āļāļāļēāļĢāđāļŠāđāļĨāļđāļāļāļĢāļ°āļŠāļļāļ āļāļĒāđāļēāļāđāļĢāļāđāļāļēāļĄ āļāļēāļĢāļāļĢāļ§āļāļāļīāļŠāļđāļāļāđāļāļĩāđāļāđāļāđāļāļāļēāļāļŦāļĨāļąāļāļāļēāļāļāļąāļāļāļĨāđāļēāļ§āļāđāļ§āļĒāļ§āļīāļāļĩāļĄāļēāļāļĢāļāļēāļāļāļąāđāļāđāļŦāđāļĢāļđāļāđāļāļāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩ āđāļāđāļāđāļ (STR profile) āļāļĩāđāđāļĄāđāļŠāļĄāļāļđāļĢāļāđāļŦāļĢāļ·āļāđāļĄāđāļŠāļēāļĄāļēāļĢāļāļāļģāđāļāđāļāđāļāļēāļĢāđāļāļāļąāđāļāļĻāļēāļĨāđāļāđ āļāļĢāļ°āļāļ§āļāļāļēāļĢāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđ (Direct PCR) āļŦāļĢāļ·āļāļāļēāļĢāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāļāļēāļāļ§āļąāļāļāļļāļāļĒāļēāļāđāļāļĒāļāļĢāļāđāļāļĒāđāļĄāđāļāđāļēāļāļāļēāļĢāļŠāļāļąāļāļāļĩāđāļāđāļāđāļāđāļāđāļāļđāļ āđāļŠāļāļāđāļŦāđāđāļŦāđāļāļ§āđāļēāļŠāļēāļĄāļēāļĢāļāđāļāļīāđāļĄāļāļ§āļēāļĄāļŠāļĄāļāļđāļĢāļāđāļāļāļāļĢāļđāļāđāļāļāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāļāļēāļāļ§āļąāļāļāļļāļāļĒāļēāļāļāļĩāđāļĄāļĩāļāļĩāđāļāđāļāđāļāļāļēāļ āļāļēāļĢāļŠāļąāļĄāļāļąāļŠ āđāļāļĒāļĄāļĩāļāļēāļāļ§āļīāļāļąāļĒāđāļāļĩāļĒāļāđāļāđāļŠāļāļāļāļēāļāļāđāļāļāļŦāļāđāļēāļāļĩāđāļāļģāļāļĢāļ°āļāļ§āļāļāļēāļĢāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđāļĄāļēāđāļāđāđāļāļ·āđāļāđāļāļīāđāļĄāļāļĢāļīāļĄāļēāļ āļāļĩāđāļāđāļāđāļāļāļēāļāļāļĢāļ°āļŠāļļāļāļāļ·āļāđāļĨāļ°āļāļĨāļāļāļāļĢāļ°āļŠāļļāļāļāļ·āļ āđāļĨāļ°āļāļąāđāļāļŠāļāļāļāļēāļāļĒāļąāļāļĄāļĩāļāļāļāđāļāļāļāļēāļĢāļ§āļīāļāļąāļĒāļāļĩāđāļāļģāļāļąāļ āļāļąāļāļāļąāđāļ āđāļ āļāļēāļāļ§āļīāļāļąāļĒāļāļĩāđāļāļēāļāļāļđāđāļ§āļīāļāļąāļĒāļāļķāļāļāļģāļāļĢāļ°āļāļ§āļāļāļēāļĢāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđāļĄāļēāđāļāđāđāļāļāļēāļĢāđāļāļīāđāļĄāļāļĢāļ°āļŠāļīāļāļāļīāļ āļēāļāđāļĨāļ°āļāļ§āļēāļĄāļŠāļģāđāļĢāđāļāđāļ āļāļēāļĢāļāļąāļāļāļģāļĢāļđāļāđāļāļāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļ āļāļĨāļāļēāļĢāļāļāļĨāļāļāđāļŠāļāļāđāļŦāđāđāļŦāđāļāļ§āđāļē āļāļēāļĢāļŠāļāļąāļāļāļĩāđāļāđāļāđāļāļĄāļĩāļāļĢāļ°āļŠāļīāļāļāļīāļ āļēāļāļāđāļģ āđāļĨāļ°āļāļģāđāļŦāđāđāļāļīāļāļāļēāļĢāļŠāļđāļāđāļŠāļĩāļĒāļāļĩāđāļāđāļāđāļāđāļāļĄāļēāļāļāļķāļāļĢāđāļāļĒāļĨāļ° 40 āļāļāļāļāļĩāđāļāđāļāđāļāļāļąāđāļāļāđāļ āđāļāļāļāļ°āļāļĩāđāļāļĢāļ°āļāļ§āļāļāļēāļĢāļĒāļīāļāļāļ·āļ āļāļģāđāļŦāđāļŠāļđāļāđāļŠāļĩāļĒāļāļĩāđāļāđāļāđāļāđāļāļāļĩāļāļĢāđāļāļĒāļĨāļ° 27 āđāļĄāļ·āđāļāļāļģāļāļēāļĢāđāļāļĢāļĩāļĒāļāđāļāļĩāļĒāļāļāļēāļĢāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāđāļāļ āļĄāļēāļāļĢāļāļēāļ āļāļēāļĢāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāđāļāļāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđ āđāļĨāļ°āļāļēāļĢāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāđāļāļāđāļāļĨāļđāļāļąāđāļ āļāļĩāļāļĩāļāļēāļĢāđ āļāļēāļāļāļĨāļāļāļāļĢāļ°āļŠāļļāļāļāļ·āļ 9 āļĄāļĄ. āļāļĩāđāļāđāļēāļāļāļēāļĢāļĒāļīāļāđāļĨāđāļ§ āļāļāļ§āđāļēāļāļēāļĢāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāđāļāļāđāļāđāļĢāđāļāļāļĩāļāļĩ āļāļēāļĢāđāļŠāļēāļĄāļēāļĢāļāđāļŦāđāļāļģāļāļ§āļāļāļąāļĨāļĨāļĩāļĨāļāļĩāđāļŠāļđāļāļāļĩāđāļŠāļļāļāđāļĄāļ·āđāļāđāļāļĩāļĒāļāļāļąāļāļāļĩāļāļŠāļāļāļ§āļīāļāļĩ āđāļāļĒāļĄāļĩāļāđāļēāđāļāļĨāļĩāđāļĒāđāļĨāļ°āļāđāļ§āļāđāļāļ·āđāļāļāļ·āļ (Credible interval) āļāļĒāļđāđāļāļĩāđ 11.1 (7.9â13.9) āļŠāļģāļŦāļĢāļąāļāļ§āļīāļāļĩāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđ 5.6 (3.0â7.7) āļŠāļģāļŦāļĢāļąāļāļ§āļīāļāļĩāļĄāļēāļāļĢāļāļēāļ āđāļĨāļ° 2.3 (0.2â4.0) āļŠāļģāļŦāļĢāļąāļāļ§āļīāļāļĩāđāļāļĨāļđāļāļąāđāļāļāļĩāļāļĩāļāļēāļĢāđ āļāļēāļĢāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāđāļāļāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđāļāļēāļāļāļĨāļāļāļāļĢāļ°āļŠāļļāļāļāļ·āļ āļāļĩāđāļāđāļēāļāļāļēāļĢāļĒāļīāļāđāļĨāđāļ§āļŠāļēāļĄāļāļāļīāļ (9 āļĄāļĄ. 7.62 āļĄāļĄ. āđāļĨāļ° 5.5 āļĄāļĄ.) āđāļŠāļāļāđāļŦāđāđāļŦāđāļāļ§āđāļēāļāļģāļāļ§āļāļāļąāļĨāļĨāļĩāļĨāļāļĩāđāđāļāđāļĢāļąāļāļāļēāļ āļāļĢāļ°āļŠāļļāļāđāļĨāļ°āļāļ·āļāļāļąāđāļāļŠāļēāļĄāļāļāļīāļāđāļĄāđāđāļāļāļāđāļēāļāļāļąāļāļāļĒāđāļēāļāļĄāļĩāļāļąāļĒāļŠāļģāļāļąāļāļāļēāļāļŠāļāļīāļāļī āļāļāļāļāļēāļāļāļĩāđ āļāļēāļĢāļāļāļĨāļāļāļāļēāļĢāļāļģāļĨāļāļāđāļŦāļāļļāļāļēāļĢāļāđāļāļĢāļīāļāļāļĩāđāļĄāļĩāļāļēāļĢāđāļāđāļāļ·āļāļĢāđāļ§āļĄāļāļąāļāđāļŠāļāļāđāļŦāđāđāļŦāđāļāļ§āđāļēāļĄāļĩāļĢāļđāļāđāļāļāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāļāļŠāļĄāļāļķāļāļĢāđāļāļĒāļĨāļ° 40 āļāļāļāļāļąāļ§āļāļĒāđāļēāļ āļāļąāļāļāļąāđāļāļāļēāļĢāđāļāļĨāļāļĨāļāļēāļāļāļāļĩāļāļ§āļēāļĄāļāļĢāļīāļāļāļķāļāļĄāļĩāļāđāļēāļāļ°āļāļ§āļēāļĄāļĒāļļāđāļāļĒāļēāļāđāļĨāļ°āļāļąāļāļāđāļāļ āļāļķāđāļāļāđāļēāļāļ°āļāđāļāļāļĄāļĩāļāļēāļĢāļāļģ āļĢāļ°āļāļāļāļēāļĢāđāļāļĨāļāļĨāļāļĩāđāđāļāđāđāļĄāđāļāļĨāļāļēāļĢāļāļģāļāļ§āļāļāđāļēāļāļ§āļēāļĄāļāđāļēāļāļ°āđāļāđāļ (Expert interpretation system using continuous model) āļĄāļēāđāļāđ āđāļāļĒāļŠāļĢāļļāļ āļāļēāļĢāļāļģāđāļāļāļāļīāļāđāļāđāļĢāđāļāļāļĩāļāļĩāļāļēāļĢāđāļĄāļēāđāļāđāđāļāļ·āđāļāļāļąāļāļāļģāļĨāļēāļĒāļāļīāļĄāļāđāļāļĩāđāļāđāļāđāļāļāļēāļāļāļĨāļāļāļāļĢāļ°āļŠāļļāļāļāļĢāļ°āļŠāļāļāļ§āļēāļĄāļŠāļģāđāļĢāđāļ āļĄāļĩāļāļĢāļ°āļŠāļīāļāļāļīāļ āļēāļāļŠāļđāļ āļĢāļ§āļāđāļĢāđāļ§ āđāļĨāļ°āļāļĢāļ°āļŦāļĒāļąāļāļāđāļēāđāļāđāļāđāļēāļĒ āđāļŦāļĄāļēāļ°āļāļąāļāļāļēāļĢāļāļģāđāļāđāļāđāļāļąāļāļāļąāļ§āļāļĒāđāļēāļāļāļĨāļāļāļāļĢāļ°āļŠāļļāļāļāļēāļāļāļāļĩāļāļ§āļēāļĄāļāļĢāļīāļāļāđāļāđ
Tiger hair morphology and its variations for wildlife forensic investigation
Tiger population has dramatically decreased due to illegal consumption and
commercialisation of their body parts. Frequently, hair samples are the only evidence
found in the crime scene. Thus, they play an important role in species identification for
wildlife forensic investigation. In this study, we provide the first in-depth report on a
variety of qualitative and quantitative characteristics of tiger guard hairs (24 hairs per
individual from four individuals). The proposed method could reduce subjectivity of
expert opinions on species identification based on hair morphology. Variations in 23 hair
morphological characteristics were quantified at three levels: hair section, body region,
and intra-species. The results indicate statistically significant variations in most
morphological characteristics in all levels. Intra-species variations of four variables,
namely hair length, hair index, scale separation and scale pattern, were low. Therefore,
identification of tiger hairs using these multiple features in combination with other
characteristics with high inter-species variations (e.g. medulla type) should bring about
objective and accurate tiger hair identification. The method used should serve as a
guideline and be further applied to other species to establish a wildlife hair morphology
database. Statistical models could then be constructed to distinguish species and provide
evidential values in terms of likelihood ratios