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  • If the unwanted objects detected by OpenComet are manually d

    2020-11-23

    If the unwanted objects detected by OpenComet are manually deleted, and then the DNA (%) in tail is computed, the performance of the present method can be compared more effectively. Therefore, performance of OpenComet is analysed in two ways as Approach 1 and Approach 2. Approach 1, considers the DNA (%) in tail of each clinical case with Comets + Outliers as indicated in the output file. In Approach 2, the wrongly detected objects are manually deleted and the program is updated for recalculating the DNA (%) in tail. The results of these two approaches are shown in Figs. 7(b) and 7(c) respectively. In regression analysis the value obtained for CometScore is 0.14 and equation of regression is . The value obtained for OpenComet with Approaches 1 and 2 are 0.64 and 0.84 respectively and the corresponding equations of regression are and respectively. While comparing the values of different methods, the proposed method gives the highest value 0.9 with as the equation of regression. The proposed method shows better performance in all the three phases: comet segmentation, partitioning and quantification, when compared with most recent related works. One limitation of the proposed method is that, while selecting the four squares for background estimation, the presence of any comets in the corner may eventually lead to tail loss in comet segmentation, due to the variation in the background value. This can be eliminated by selecting an area where no comets are present. A second limitation is the computation time required for image analysis. The average time required to analyse an image using the proposed method is 8 s whereas it is 2 s using OpenComet [12]. The testing was done on Intel Core i5-3210M CPU @2.50 GHz with 8 GB RAM.
    Conclusion
    Introduction Staining cl 2 in SDS-PAGE gels is a well-established method with a range of stains available. Coomassie brilliant blue R250 is arguably the most widely used staining method. Colorimetric methods are popular due to their simplicity, although they do require lengthy destaining steps, with some alternatives to Coomassie including Amido black and direct red 81 stains [1]. Colloidal stains such as silver [2] are the most sensitive but require multiple incubation and wash steps.
    Materials and methods
    Results
    Discussion
    Acknowledgements This research was funded by the South African Medical Research Council, National Research Foundation and the University of KwaZulu-Natal Research Incentive fund.
    Introduction Most forensic laboratories have experienced the inability to detect or amplify DNA from moist biological stains or reference samples on cotton buds, which had been stored and/or shipped at soaking wet conditions. It has been shown that the inactivation rate of a protein (acid phosphatase) in semen stains is exponentially related to the relative humidity, the rate being 1,000,000-fold higher at 100% humidity than at 40% [1]. The degradation of DNA often parallels the degradation of proteins, and many of the processes that degrade DNA depend on the presence of water [2], [3]. Therefore, we expected that DNA in biological stains and reference samples would show a rapid decay at high relative humidity. To see if this was true, stains of whole blood or buccal cells were incubated at various conditions of relative humidity and temperature. The quality and quantity of the remaining DNA was assessed by PCR.
    Methods and materials Whole blood (5 μl) or buccal cells were spotted onto pieces (4×4 mm) of Whatman filter paper No. 3 and air-dried overnight. The stains were incubated in closed boxes at 0%, 50%, 80% and 100% relative humidity at room temperature, 35, 45, 55 and 65 °C. Constant humidity was maintained by the inclusion of H2O, saturated solutions of (NH4)2SO4 (80% humidity), NaHSO4, H2O (50% humidity) or silica gel. One series of bloodstains on microscopic slides was transferred to soaking wet (Milli-Q grade water) cotton buds and incubated at 100% humidity at room temperature. DNA was extracted from the stains using QIAamp® DNA Mini Kit (Qiagen, Germany). PCR of a 1600 bp segment was performed using primers targeting the human ACP1 locus from intron 4 to exon 5 and a 273 bp segment was amplified using primers targeting exon 4 at the human HFE-locus. Real time PCR was performed using ABI-Prism-7000 SDS (Applied Biosystems) and primers and probe targeting a 147 bp segment in exon 4 of the human HFE-gene.