Low-volume amplification on chemically structured chips using the PowerPlex16 DNA amplification kit

University of Freiburg, Freiburg, Baden-Württemberg, Germany
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin (Impact Factor: 2.71). 02/2006; 120(1):42-8. DOI: 10.1007/s00414-005-0041-2
Source: PubMed


In forensic DNA analysis, improvement of DNA typing technologies has always been an issue. It has been shown that DNA amplification in low volumes is a suitable way to enhance the sensitivity and efficiency of amplification. In this study, DNA amplification was performed on a flat, chemically structured glass slide in 1-microl reaction volumes from cell line DNA contents between 1,000 and 4 pg. On-chip DNA amplification reproducibly yielded full allelic profiles from as little as 32 pg of template DNA. Applicability on the simultaneous amplification of 15 short tandem repeats and of a segment of the Amelogenin gene, which are routinely used in forensic DNA analysis, is shown. The results are compared to conventional in-tube amplification carried out in 25-microl reaction volumes.

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    • "The most widely used, single-tube, DNA extraction method after LCM is the method based on proteinase K. Another recently developed technology (AmpliGrid slide, Advalytix AG, Germany) uses chemically structured microscope slides designed for ultra-low-volume applications . After the laser microdissection of cells (even of a single cell) into one of the hydrophilic reaction sites on the slide, DNA extraction and PCR can be performed directly on these reaction sites (Schmidt et al. 2006; Vandewoestyne and Deforce 2010). The combination of the AmpliGrid technology with micromanipulation has been already successfully applied by Li et al. (2009a) for a forensic analysis of different types of material evidence (Li et al. 2009b). "
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    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 · 1.48 Impact Factor
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    • "In fact, Miyazaki et al. [9] demonstrated complete haploid-type electropherograms from a single sperm following nuclear DNA amplification using improved primer extension preamplification polymerase chain reaction (I-PEP-PCR). Together, these studies support reports indicating the high sensitivity of on chip LV-PCR [8], [10], [11]. "
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    ABSTRACT: Genetic profiling of sperm from complex biological mixtures such as sexual assault casework samples requires isolation of a pure sperm population and the ability to analyze low abundant samples. Current standard procedure for sperm isolation includes preferential lysis of epithelial contaminants followed by collection of intact sperm by centrifugation. While effective for samples where sperm are abundant, this method is less effective when samples contain few spermatozoa. Laser capture microdissection (LCM) is a proven method for the isolation of cells biological mixtures, even when found in low abundance. Here, we demonstrate the efficacy of LCM coupled with on-chip low volume PCR (LV-PCR) for the isolation and genotyping of low abundance sperm samples. Our results indicate that this method can obtain complete profiles (13-16 loci) from as few as 15 sperm cells with 80% reproducibility, whereas at least 40 sperm cells are required to profile 13-16 loci by standard 'in-tube' PCR. Further, LCM and LV-PCR of a sexual assault casework sample generated a DNA genotype that was consistent with that of the suspect. This method was unable, however, to analyze a casework sample from a gang rape case in which two or more sperm contributors were in a mixed population. The results indicate that LCM and LV-PCR is sensitive and effective for genotyping sperm from sperm/epithelial cell mixtures when epithelial lysis may be insufficient due to low abundance of sperm; LCM and LV-PCR, however, failed in a casework sample when spermatozoa from multiple donors was present, indicating that further study is necessitated.
    PLoS ONE 08/2011; 6(8):e22316. DOI:10.1371/journal.pone.0022316 · 3.23 Impact Factor
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    • "Mithilfe der verwendeten Mikrokugeln gelingt die Zellaufnahme von Glas, Metall und Kunststoff. Der Transfer der Kugel mitsamt anhaftender Zelle erfolgt entweder in ein PCR-Gefäß (MicroAmp Reaction Tube with Cap; 0,2 ml; ABI) oder auf den Objektträger AmpliGrid® (Advalytix) [10] [11] "
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    ABSTRACT: Mithilfe eines Mikroskops mit einem digitalen Mikromanipulator werden einzelne Epithelzellen von Spurenträgern gesichert. Die forensische DNA-Analyse der Einzelzellen erfolgt mittels Sequenzierung der hypervariablen Region der mtDNA. With a technique using a microscope with a digital micromanipulator, single epithelial cells can be lifted off the surface of material evidence and can be directly made available for forensic DNA-testing. DNA-testing is then accomplished by sequencing the hypervariable region of the mtDNA.
    BioSpektrum 03/2011; 17(2):185-187. DOI:10.1007/s12268-011-0029-z
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