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Wasserstrom A.,Nucleix Ltd. | Frumkin D.,Nucleix Ltd. | Davidson A.,Nucleix Ltd. | Shpitzen M.,DNA and Forensic Biology Laboratory | And 2 more authors.
Forensic Science International: Genetics | Year: 2013

Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This sample likely represents true semen because sperm cells were detected from an adjacent sample from the same garment, therefore in this case the assay appears to be more sensitive than the microscopic examination. These results demonstrate that this assay is a bona fide confirmatory test for semen. © 2012 Elsevier Ireland Ltd.


Frumkin D.,Nucleix Ltd. | Wasserstrom A.,Nucleix Ltd. | Budowle B.,University of North Texas Health Science Center | Davidson A.,Nucleix Ltd.
Forensic Science International: Genetics | Year: 2011

Identifying the source tissue of biological material found at crime scenes can be very informative in a number of cases. Despite their usefulness, current visual, catalytic, enzymatic, and immunologic tests for presumptive and confirmatory tissue identification are applicable only to a subset of samples, might suffer limitations such as low specificity, lack of sensitivity, and are substantially impacted by environmental insults. Moreover these assays are incompatible and thus cannot be multiplexed. Thus they are less amenable to automation. In addition their results are operator-dependent. A better alternative approach is tissue identification based on messenger RNA (mRNA) or microRNA (miRNA); however, RNA is not as stable as DNA, and requires the use of non-standard procedures by forensic laboratories. Herein a DNA-based assay is described that enables tissue identification based on detection of tissue-specific methylation patterns. DNA samples are subjected to digestion by a methylation-sensitive restriction endonuclease followed by multiplex amplification of specific genomic targets with fluorescent-labeled primers, capillary electrophoresis of amplification products, and automatic signal analysis by dedicated software, yielding the source tissue of the sample. The single tube assay was designed for easy integration by forensic laboratories (as the assay utilizes the same platforms as current forensic STR profiling). The system is fully automatable, provides operator-independent results, and allows combining tissue identification with profiling in a single procedure. The assay was tested on 50 DNA samples from blood, saliva, semen, and skin epidermis, and the source tissue was successfully identified in all cases. Detection of semen and DNA profiling were combined into one assay and the ability to detect mixtures of semen and saliva in various ratios was demonstrated. The assay correctly detected semen in all samples where it was present, and the calculated percentage of semen was comparable to the fraction of semen in the samples. The results demonstrate that methylation-based tissue identification is more than a proof-of-concept. The methodology holds promise as another viable forensic DNA analysis tool for characterization of biological materials. © 2010 Elsevier Ireland Ltd.


Frumkin D.,Nucleix Ltd. | Wasserstrom A.,Nucleix Ltd. | Davidson A.,Nucleix Ltd. | Grafit A.,Serious Crime Unit Mobile Laboratory
Forensic Science International: Genetics | Year: 2010

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus® yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system. © 2009 Elsevier Ireland Ltd. All rights reserved.


Uziel O.,Tel Aviv University | Yerushalmi R.,Tel Aviv University | Zuriano L.,Tel Aviv University | Naser S.,Tel Aviv University | And 16 more authors.
Oncotarget | Year: 2016

BRCA1 mutation is associated with carcinogenesis, especially of breast tissue. Telomere maintenance is crucial for malignant transformation. Being a part of the DNA repair machinery, BRCA1 may be implicated in telomere biology. We explored the role of BRCA1 in telomere maintenance in lymphocytes of BRCA1/2 mutation carriers and in in vitro system by knocking down its expression in non-malignant breast epithelial cells. The results in both systems were similar. BRCA1/2 mutation caused perturbation of telomere homeostasis, shortening of the single stranded telomere overhang and increased the intercellular telomere length variability as well as the number of telomere free chromosomal ends and telomeric circles. These changes resulted in an increased DNA damage status. Telomerase activity, inducibility and expression remained unchanged. BRCA1 mutation resulted also in changes in the binding of shelterin proteins to telomeres. DNMT-1 levels were markedly reduced both in the carriers and in in vitro system. The methylation pattern of the sub-telomeric regions in carriers suggested hypomethylation in chromosome 10. The expression of a distinct set of genes was also changed, some of which may relate to pre-disposition to malignancy. These results show that BRCA gene products have a role in telomere length homeostasis. It is plausible that these perturbations contribute to malignant transformation in BRCA mutants.


Grant
Agency: European Commission | Branch: H2020 | Program: SME-1 | Phase: PHC-12-2015-1 | Award Amount: 71.43K | Year: 2015

Nucleix developed a revolutionary platform for the development of Methylation Bio-markers and Bio-Marker panels for screening (early detection of disease) and diagnosis based on epigenetic markers. The platform identifies changes in specific methylation locations of DNA extracted from body fluids such as urine, blood, stools and sputum. A Bio-Markers panel goal is to distinguish between sick and healthy individuals. The companys expertise include the identification of the specific loci, comprising a panel of markers that can work in multiplex and generate highly accurate test. Nucleix technology was proven to be extremely sensitive and specific in its first application. Nucleix focuses initially on the field of cancer detection and screening. Cancer cost the EU 126 billion in 2009, which was 1.07% of EU GDP. 2.45m People in the EU were diagnosed with cancer in 2008, when 1.23 million people in the EU died from cancer in 2008. This is one of the most significant public health issues in the world and in the EU. Nucleix first product, Bladder EpiCheck is a urine test for the detection of Bladder Cancer. It is aimed for monitoring bladder cancer patients as this is a disease with high recurrence rate and requires tight surveillance. This product will be launched in Q4 of 2015. The test is based on a panel of 15 non-overlapping Methylation markers at loci that were identified, developed and owned by the company. This product which has better sensitivity and specificity than any other non-invasive test is a proof of the companys ability to translate its technology to applicable products. The company plans to develop EPICHECK - diagnostic kits using multi-loci panel of markers, for the detection and screening of lung cancer and colorectal cancer in blood samples which is the subject of this applications. EPICHECK will be launched rapidly, and will provide revenues of over 320m Euro in its 5th year of commercial activities.


PubMed | Nucleix Ltd.
Type: Journal Article | Journal: Forensic science international. Genetics | Year: 2010

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.


PubMed | Nucleix Ltd.
Type: Journal Article | Journal: Forensic science international. Genetics | Year: 2012

Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assays applicability in real-life situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This sample likely represents true semen because sperm cells were detected from an adjacent sample from the same garment, therefore in this case the assay appears to be more sensitive than the microscopic examination. These results demonstrate that this assay is a bona fide confirmatory test for semen.


PubMed | Nucleix Ltd.
Type: Journal Article | Journal: Forensic science international. Genetics | Year: 2011

Identifying the source tissue of biological material found at crime scenes can be very informative in a number of cases. Despite their usefulness, current visual, catalytic, enzymatic, and immunologic tests for presumptive and confirmatory tissue identification are applicable only to a subset of samples, might suffer limitations such as low specificity, lack of sensitivity, and are substantially impacted by environmental insults. Moreover these assays are incompatible and thus cannot be multiplexed. Thus they are less amenable to automation. In addition their results are operator-dependent. A better alternative approach is tissue identification based on messenger RNA (mRNA) or microRNA (miRNA); however, RNA is not as stable as DNA, and requires the use of non-standard procedures by forensic laboratories. Herein a DNA-based assay is described that enables tissue identification based on detection of tissue-specific methylation patterns. DNA samples are subjected to digestion by a methylation-sensitive restriction endonuclease followed by multiplex amplification of specific genomic targets with fluorescent-labeled primers, capillary electrophoresis of amplification products, and automatic signal analysis by dedicated software, yielding the source tissue of the sample. The single tube assay was designed for easy integration by forensic laboratories (as the assay utilizes the same platforms as current forensic STR profiling). The system is fully automatable, provides operator-independent results, and allows combining tissue identification with profiling in a single procedure. The assay was tested on 50 DNA samples from blood, saliva, semen, and skin epidermis, and the source tissue was successfully identified in all cases. Detection of semen and DNA profiling were combined into one assay and the ability to detect mixtures of semen and saliva in various ratios was demonstrated. The assay correctly detected semen in all samples where it was present, and the calculated percentage of semen was comparable to the fraction of semen in the samples. The results demonstrate that methylation-based tissue identification is more than a proof-of-concept. The methodology holds promise as another viable forensic DNA analysis tool for characterization of biological materials.

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