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Guleria S.,Dr. Y.S. Parmar University of Horticulture and Forestry | Walia A.,DNA Unit | Chauhan A.,Dr. Y.S. Parmar University of Horticulture and Forestry | Shirkot C.K.,Dr. Y.S. Parmar University of Horticulture and Forestry
Proceedings of the National Academy of Sciences India Section B - Biological Sciences | Year: 2016

Rhizosphere of apple trees of Trans Himalayas is great niche of proteolytic bacteria which may have potential for use in industries. A total of 76 proteolytic bacteria were obtained from rhizosphere soil and root endosphere of apple trees from different sites of four locations viz., Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh, India, which were screened for plant growth promoting traits including production of indole acetic acid, phosphate solubilization, hydrogen cyanide production and production of antifungal metabolite. Among them, 17 isolates that exhibited maximum alkaline protease activity were selected. The diversity elucidation and differentiation of isolates was achieved through their biochemical characterization and amplified ribosomal DNA restriction analysis with three tetra-cutter restriction enzymes (HpaII, HinfI and TaqI), the representative cluster groups were identified by 16S rDNA sequencing. This brings to light the predominance of Bacillus sp. and presence of remarkable functional and genotypic intraspecific diversity in apple rhizosphere. Proteolytic activity among selected isolates ranged between 50 and 2,770 µg/ml/min at pH 8.0 and the maximum was observed for Bacillus amyloliquefaciens (2,770 µg/ml/min) followed by B. subtilis (1,300.97 µg/ml/min). Bacillus amyloliquifaciens in particular showed great potential of alkaline protease production which agrees its exploitation in detergent industry. To the best of authors’ knowledge, this is first report for genetic and functional diversity of alkaline protease producing PGPR associated with apple rhizosphere in Trans-Himalayan region of Himachal Pradesh. Additionally the generated information may serve as a base line data for developing effective alkaline protease preparation for the industrial use. © 2014, The National Academy of Sciences, India. Source


LaSalle H.E.,DNA Unit | Duncan G.,DNA Unit | McCord B.,Florida International University
Forensic Science International: Genetics | Year: 2011

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler® Duo, utilizes a TaqMan® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user. © 2010 Elsevier Ireland Ltd. Source


Simson Oechsle C.,DNA Unit | Simson Oechsle C.,Boston University | Simson Oechsle C.,DNA Labs International | Haddad S.,DNA Unit | And 4 more authors.
Journal of Forensic Sciences | Year: 2014

Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 μL of saliva for the RSID™ Saliva cards, 0.03 μL of saliva for Quantifiler® Duo, and 0.001 μL of semen for Quantifiler® Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection. © 2013 American Academy of Forensic Sciences. Source


Balamurugan K.,University of Southern Mississippi | Bombardi R.,University of Southern Mississippi | Duncan G.,DNA Unit | Mccord B.,Florida International University
Electrophoresis | Year: 2014

The focus of this study is to evaluate the application of epigenetic markers as a forensic tool for the determination of semen present in sexual assault cases. A series of genetic loci were screened in order to identify certain epigenetic markers displaying differential methylation that can allow semen to be differentiated from blood, buccal cells, skin epidermis, and vaginal epithelial cells. Of the different loci tested, a panel of six markers, DACT1, USP49, DDX4, Hs_INSL6_03, Hs_ZC3H12D_05, and B_SPTB_03 were identified to contain tissue-specific differential methylation. Samples ranging from 9-21 for each tissue type were collected and subjected to bisulfite modification. The bisulfite modified DNA was amplified by PCR, and analyzed by pyrosequencing to quantitate the level of methylation at each marker. All six markers successfully differentiated semen samples from the other four tissue types analyzed. Sperm DNA was hypomethylated in all but one marker, B_SPTB_03, where this marker showed hypermethylation. Mean methylation percentages for semen samples were statistically significant from mean methylation percentages of the other four tissues studied (p < 0.01). The results of this study demonstrate the applicability of epigenetic markers as a novel tool for determination of spermatozoa and to identify the tissue source of origin of a DNA sample. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Duchamp V.,University of Cologne | Duchamp V.,DNA Unit | Bastisch I.,DNA Unit | Schneider P.M.,University of Cologne
Forensic Science International: Genetics Supplement Series | Year: 2015

Two statistical models have been developed to interpret locus drop-out resulting from degraded DNA samples. These models are based on an experimental dataset of 600 genotyping experiments. The first model presents the probability of locus drop-out against the average peak height with a lower and upper confidence interval. For a given peak height average, the probability of drop-out can be quite wide. This probability decreases rapidly when the profile quality increases. For the second model, we have fitted the probability of locus drop-out against the number of base pairs with a lower and upper confidence interval. The probability of drop-out is increasing along with the number of base pairs. Despite DNA degradation effects, the drop-out probability does not get higher than 0.5. © 2015 Elsevier Ireland Ltd. Source

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