Digoxigenin labeling and laser capture microdissection of male cells
ABSTRACT Laser capture microdissection (LMD) is a relatively new technique for the isolation of single cells. The application in forensic investigations has become more and more widespread, especially to select spermatozoa out of mixtures with vaginal cells. In particular in cases with low numbers of sperm it could be profitable to isolate all male cells (e.g. sperm and male epithelial cells) instead of focussing on the sperm only. Therefore, the specific labelling and detection of the male cells in a male/female cell mixture is necessary. In order to label all cells carrying a Y-chromosome we used a digoxigenin labelled chromosome Y hybridisation probe (Q Biogen). The stained cells were isolated with the SL microCut LMD system from Molecular Machines & Industries AG (MMI). At least ten diploid male cells were required to obtain a partial STR profile, with 20 cells, a full profile could be obtained.
- SourceAvailable from: Barbara Jarząb
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- "Fluorescence in situ hybridisation (FISH) probes, specific for X and Y chromosomes, are used to distinguish male from female cells. This application becomes widespread in forensic investigations of sexual assault cases with low numbers of sperm cells or male/female mixtures of blood or epithelial cells (Anslinger et al. 2005; Vandewoestyne and Deforce 2010). In medical sciences, LCM is used in cancer research to determine the cell-specific gene expression profile from heterogeneous human tissues (Curran et al. 2000; Cho-Vega et al. 2005). "
ABSTRACT: This review presents the basic problems and currently available molecular techniques used for genetic profiling in disaster victim identification (DVI). The environmental conditions of a mass disaster often result in severe fragmentation, decomposition and intermixing of the remains of victims. In such cases, traditional identification based on the anthropological and physical characteristics of the victims is frequently inconclusive. This is the reason why DNA profiling became the gold standard for victim identification in mass-casualty incidents (MCIs) or any forensic cases where human remains are highly fragmented and/or degraded beyond recognition. The review provides general information about the sources of genetic material for DNA profiling, the genetic markers routinely used during genetic profiling (STR markers, mtDNA and single-nucleotide polymorphisms [SNP]) and the basic statistical approaches used in DNA-based disaster victim identification. Automated technological platforms that allow the simultaneous analysis of a multitude of genetic markers used in genetic identification (oligonucleotide microarray techniques and next-generation sequencing) are also presented. Forensic and population databases containing information on human variability, routinely used for statistical analyses, are discussed. The final part of this review is focused on recent developments, which offer particularly promising tools for forensic applications (mRNA analysis, transcriptome variation in individuals/populations and genetic profiling of specific cells separated from mixtures).Journal of applied genetics 02/2012; 53(1):41-60. DOI:10.1007/s13353-011-0068-7 · 1.90 Impact Factor
Forensic Medicine - From Old Problems to New Challenges, 09/2011; , ISBN: 978-953-307-262-3
- "The different cell types can be recognized by morphological characteristics, various chemical staining or fluorescence labeling techniques. These methods allow to establish a clear DNA profile from few cells present in a mixture samples that otherwise had not be detected while swabbed by the major component and not detectable in the profile ( Elliott et al., 2003; Anslinger et al., 2005; Anoruo et al., 2007 ; Sanders et al., 2006). With laser micro dissection techniques ( Anslinger et al., 2007; Vandewoestyne et al., 2009), it has been shown that cells derived from a male contributor can be analyzed separately from those derived from a female contributor after morphological or fluorescent labeling identification. "
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- "Similarly, specific detection and isolation of male cells in male/female mixtures such as fingernail scrapings, bite or licking traces and male/female blood mixtures could be a major advantage. Therefore, several groups developed staining methods to perform sex-specific labelling of cells for LCM [37–41]. To distinguish male from female cells, fluorescence in situ hybridization (FISH) is performed using Y chromosome specific probes. "
ABSTRACT: In forensic sciences, short tandem repeat (STR) analysis has become the prime tool for DNA-based identification of the donor(s) of biological stains and/or traces. Many traces, however, contain cells and, hence, DNA, from more than a single individual, giving rise to mixed genotypes and the subsequent difficulties in interpreting the results. An even more challenging situation occurs when cells of a victim are much more abundant than the cells of the perpetrator. Therefore, the forensic community seeks to improve cell-separation methods in order to generate single-donor cell populations from a mixed trace in order to facilitate DNA typing and identification. Laser capture microdissection (LCM) offers a valuable tool for precise separation of specific cells. This review summarises all possible forensic applications of LCM, gives an overview of the staining and detection options, including automated detection and retrieval of cells of interest, and reviews the DNA extraction protocols compatible with LCM of cells from forensic samples.Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 11/2010; 124(6):513-21. DOI:10.1007/s00414-010-0499-4 · 2.60 Impact Factor