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Hedman J.,Swedish National Laboratory of Forensic Science SKL | Hedman J.,Lund University | Radstrom P.,Lund University
Methods in Molecular Biology | Year: 2013

PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR analysis. © 2013 Springer Science+Business Media, LLC. Source


Dufva C.,Swedish National Laboratory of Forensic Science SKL | Nilsson A.,Swedish National Laboratory of Forensic Science SKL
Forensic Science International: Genetics Supplement Series | Year: 2011

There is often a demand for statistics to evaluate and improve different analytical methods. This is especially important in forensic DNA analysis, facing a wide variety of items and substrates, from which samples are collected. For LT DNA analysis it is valuable to have data showing what kind of results that are received, readily divided into different categories of items. We have developed a database showing the success rates for different items. During the years 2008-2010 LT DNA results were collected in 417 cases, with a success rate of 38%. Corresponding success rate per sample in the different categories of items (i.e. knives, electronics and bombs, letters and envelopes) vary between 7% and 19%. © 2011 Elsevier Ireland Ltd. Source


Hedman J.,Swedish National Laboratory of Forensic Science SKL | Hedman J.,Lund University | Dufva C.,Swedish National Laboratory of Forensic Science SKL | Noren L.,Swedish National Laboratory of Forensic Science SKL | And 4 more authors.
Forensic Science International: Genetics Supplement Series | Year: 2011

Crime scene samples often contain extraneous compounds that may interfere with PCR-based DNA analysis, resulting in imbalanced, partial or blank/negative DNA profiles. Customising the chemical content of the PCR reaction is a strategy that may increase PCR inhibitor tolerance without manipulating the sample. We have validated a modified version of Amp. FlSTR SGM Plus, replacing Ampli. Taq Gold DNA polymerase with a customised blend of two alternative polymerases, Ex. Taq Hot Start and PicoMaxx High Fidelity. Allele calls are identical to standard analysis. Stutter sizes and balance values are indistinguishable. The modified chemistry provides increased resistance to PCR inhibitors, resulting in an elevated number of detected alleles for various problematic crime scene samples. © 2011 Elsevier Ireland Ltd. Source


Albinsson L.,Swedish National Laboratory of Forensic Science SKL | Noren L.,Swedish National Laboratory of Forensic Science SKL | Hedell R.,Swedish National Laboratory of Forensic Science SKL | Ansell R.,Swedish National Laboratory of Forensic Science SKL | Ansell R.,Linkoping University
Forensic Science International: Genetics | Year: 2011

The European Standard Set of loci (ESS) has been extended with five additional short tandem repeat (STR) loci following the recommendations of the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP) to increase the number of loci routinely used by the European forensic community. Subsequently, a new extended Swedish population database, based on 425 individuals, has been assembled using the new STR multiplex kits commercially available. Allele frequencies and statistical parameters of forensic interest for 15 autosomal STR loci (D3S1358, TH01, D21S11, D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, vWA, D8S1179, FGA, D2S441, D12S391 and D19S433) were obtained from the analysis of the PowerPlex® ESX 16 System kit (Promega Corporation, USA). According to the data no evidence of deviations from Hardy-Weinberg equilibrium was found. The observed heterozygosity varies between 0.755 (TH01) and 0.892 (D1S1656). The power of discrimination was smallest for D22S1045 (0.869) and largest for D1S1656 (0.982) while the power of exclusion was smallest for TH01 (0.518) and largest for D1S1656 (0.778). A concordance study was performed on the five amplification systems: PowerPlex® ESX 16 System, PowerPlex® ESI 16 System (Promega Corporation, USA), AmpFlSTR® NGM™, AmpFlSTR® SGM Plus™ (Applied Biosystems, USA) and Investigator ESSplex (Qiagen, Germany) to reveal null alleles and other divergences between the kits. For the 425 DNA profiles included, AmpFlSTR® NGM™ revealed two null alleles, AmpFlSTR® SGM Plus™ revealed one, and Investigator ESSplex revealed a micro-variant, while the rest of the alleles showed full concordance between the kits tested. © 2010 Elsevier Ireland Ltd. Source


Hedman J.,Swedish National Laboratory of Forensic Science SKL | Hedman J.,Lund University | Dalin E.,Swedish National Laboratory of Forensic Science SKL | Rasmusson B.,Swedish National Laboratory of Forensic Science SKL | And 2 more authors.
Forensic Science International: Genetics | Year: 2011

Amylase testing has been used as a presumptive test for crime scene saliva for over three decades, mainly to locate saliva stains on surfaces. We have developed a saliva screening application for crime scene trace swabs, utilising an amylase sensitive paper (Phadebas® Forensic Press test). Positive results were obtained for all tested dried saliva stains (0.5-32 μL) with high or intermediate amylase activity (840 and 290 kU/L). Results were typically obtained within 5 min, and all samples that produced DNA profiles were positive. However, salivary amylase activities, as well as DNA concentrations, vary significantly between individuals. We show that there is no correlation between amylase activity and amount of DNA in fresh saliva. Even so, a positive amylase result indicates presence of saliva, and thereby presence of DNA. Amylase testing may be useful for screening in investigations where the number of DNA analyses is limited due to cost, e.g., in volume crime. © 2010 Elsevier Ireland Ltd. Source

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