In the autumn of 2010 SKL performed in-house validation of PowerPlex ESX 16 System (Promega). As the validation showed that very low amounts of DNA (∼10 pg) may provide correct allele callings (peaks above 50 rfu), we investigated the linear range, i.e., the interval of DNA amounts where a profile is well balanced and does not contain drop-outs and/or drop-ins. The linear range as indicated by our results is approximately from 0.5 ng (manufacturer's recommendation) to 2.0 ng of DNA. As minute DNA amounts may be detected using the kit, extra care needs to be taken not to report a contaminant allele as a part of the correct profile. A way to verify the correctness of a single donor profile in routine analysis, without using duplicate analysis, is to use conservative peak height thresholds. We determine STR marker specific peak height thresholds for each new lot of DNA profiling kits, based on the results from three different tests: heterozygote balance, signal intensity and repeatability, and PCR inhibitor tolerance. The tests also serve to verify the quality of the kit lot. Generally, the peak height thresholds vary between 200 and 250 rfu for heterozygote alleles, with doubled values used for homozygotes.

In the autumn of 2010 SKL performed in-house validation of PowerPlex ESX 16 System (Promega). As the validationshowed that very low amounts of DNA (< 10 pg) may provide correct allele callings (peaks above 50 rfu),we investigated the linear range, i.e., the interval of DNA amounts where a profile is well balanced and doesnot contain drop-outs and/or drop-ins. The linear range as indicated by our results is approximately from 0.5ng (manufacturer’s recommendation) to 2.0 ng of DNA. Profiles generated by less than 0.5 ng contained intralocus imbalances and/or drop-outs. Above 2.0 ng “bleed through” occurs due to overload of template-DNA.A way to verify the correctness of a profile, without knowing anything about the condition of the template-DNA, is to use peak height thresholds adjusted to each marker and batch of kits used. SKL performs a qualitycontrol and adjust thresholds for each batch of kits. Three main tests are performed; detection limit, inhibitortolerance and signal repeatability. The detection limit is examined to identify at which concentration intralocus imbalances and drop-outs start to increase. The ability to overcome inhibition is checked by analysingvarying amounts of blood extracted with Chelex. Finally a set of replicates of control DNA is amplified (0.5 ngtemplate-DNA) to calculate the mean peak height and standard deviation at each locus. Generally, the peakheight thresholds vary between 200 and 250 rfu for heterozygote peaks. To verify allelic peaks below the setpeak height thresholds, SKL uses consensus analysis.

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.

The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.

In this work we present a procedure for contamination monitoring in a trace search and recovery area and agraphical classification model. The recent launch of more sensitive and robust amplification kits increases thepossibility to detect minute amounts of trace DNA. As a consequence this enhances our need to establish eliminationdatabases and demands for an increased awareness on how to avoid contamination. DNA contaminatingthe evidence somewhere along the forensic process has the potential to destroy the evidence or totally confuseand mislead the crime investigation. In the forensic laboratory specific areas are designated for different partsof the process: trace search and recovery, pre-PCR, post-PCR etc. Work procedures and cleaning routines areadapted to minimise the risk of contamination. Monitoring presence of DNA in the laboratory environment, onspecific surfaces or instruments of interest, is one way to assess these risks and will in addition increase ourknowledge on how to improve cleaning routines and behaviour in the lab. A monitoring process needs to someextent be standardised in order to become unbiased and independent on an individual level, regarding bothwhere and how samples are taken and how the results are classified. The graphical model constitutes a lineartransformation of a three-dimensional “credit system” based on alleles, markers and peak heights, into a twodimensional classification. The standardisation allows results to be compared over time, and if applied to otherwork-areas comparison between different parts of the process will be possible.

In forensic DNA casework saliva stains with epithelial cells can be very useful even presenting the key evidence.Tests for amylase activity, like Phadebas® Press test, help locate stains and indicate presence of saliva. Sensitivityis high, with positive amylase tests obtained prior to detectable levels of DNA and saliva diluted to 1:100readily generate a positive reaction with Phadebas® Press test for presence of amylase. The salivary amylaseactivity varies on individual basis over time as well as it does between individuals. In addition some individualssecrete high levels of amylases [1,2]. However, amylases are present in other body fluids as well, generally toomuch lower levels than saliva. Due to sensitivity of amylase tests there is a potential interference by otherfluids when using them to verify the presence of saliva. Other studies also demonstrate that e.g. faeces can givepositive reactions.For underwear the presence of several different body fluids might have natural causes, including vaginal secretions,(menstrual) blood, urine, faeces, as well as semen and saliva. Here we present the use of Phadebas® Presstest on underwear with naturally deposited body fluids and single source body fluid mock samples including oneindividual with higher levels of amylase activity. Our results and implications are discussed.

[1] J. Hedman, E. Dalin, B. Rasmusson, R. Ansell (2011). Forensic Science International; Genetics, 5, 194–198.

[2] J. Hedman, K. Gustavsson, R. Ansell (2008). Forensic Science International; Genetics Supplement Series, 1(1), 430–432.

In cases of sexual assault, physical evidence can be of crucial importance for a conviction. Intimate samples initially collected by a physician can prove to be the only supporting evidence for the prosecution to present at court proceedings. New analysis techniques and methods have increased the positive outcome of the samples collected. This in combination with increased use of national criminal DNA databases results in the solving of sexual crimes with unknown perpetrators. The use of standardised rape care kits facilitates the work of the physician in performing an adequate sampling procedure. The Swedish "rape care kit" has been developed and updated in response to experience gained and new possibilities.

This is a genetic study of flowering time in cultivated barley with the aim to identify the alleles contributing to rapid flowering and frost resistance. We have genotyped a collection of 23 historic barley varieties for the crucial genes [VRN-1, VRN-2, VRN-3 (HvFT), Ppd-H1, CO, and Vrs1]. We have amplified the polymorphic mutations by PCR-based methods, and sequenced them to identify possible haplotype groups. The row type was not determined of all accessions, but all the Scandinavian varieties were found to carry mutant alleles of Vrs1, that indicates them to be six-row barleys. The deletion of the crucial segment of VRN-1 vernalization contributes dominant spring growth habit. We found haplotype groups 2 and 4 to be dominant in Northern barleys whereas haplotype groups 1 and 5 dominated in south. The presence of dominant allele VRN-2 gene is addressed to floral repression until plants get vernalized. Most of the 23 varieties were found to have deleted allele of VRN-2, which is connected with a spring growth habit. The only four of the accessions that have the dominant allele of Ppd-H1 that contribute flowering are generally from the south of Europe. HvFT and CO genes CO-interact to influence flowering time. CO haplotype grouping suggest a geographical distribution of different alleles but needs more disseminations. Certain HvFT alleles cause extremely early flowering during apex development in the varieties that have deletion of VRN-2 alleles under long days. VRN-3 alleles of 14 varieties were identified.

The development of agriculture is closely associated with the domestication of wheat, one of the earliest crop species. During domestication key genes underlying traits important to Neolithic agriculture were targeted by selection. One gene believed to be such a domestication gene is NAM-B1, affecting both nutritional quality and yield but with opposite effects. A null mutation, first arisen in emmer wheat, decreases the nutritional quality but delays maturity and increases grain size; previously the ancestral allele was believed lost during the domestication of durum and bread wheat by indirect selection for larger grain. By genotyping 63 historical seed samples originating from the 1862 International Exhibition in London, we found that the ancestral allele was present in two spelt wheat and two bread wheat cultivars widely cultivated at the time. This suggests that fixation of the mutated allele of NAM-B1 in bread wheat, if at all, occurred during modern crop improvement rather than during domestication. We also discuss the value of using archaeological and historical plant material to further the understanding of the development of agriculture.

Wheat breeding during the 20th century has put large efforts into reducing straw length and increasing harvest index. In the 1920s an allele of Rht8 with dwarfing effects, found in the Japanese cultivar “Akakomugi,” was bred into European cultivars and subsequently spread over the world. Rht8 has not been cloned, but the microsatellite marker WMS261 has been shown to be closely linked to it and is commonly used for genotyping Rht8. The “Akakomugi” allele is strongly associated with WMS261-192bp. Numerous screens of wheat cultivars with different geographical origin have been performed to study the spread and influence of the WMS261-192bp during 20th century plant breeding. However, the allelic diversity of WMS261 in wheat cultivars before modern plant breeding and introduction of the Japanese dwarfing genes is largely unknown. Here, we report a study of WMS261 allelic diversity in a historical wheat collection from 1865 representing worldwide major wheats at the time. The majority carried the previously reported 164 bp or 174 bp allele, but with little geographical correlation. In a few lines, a rare 182 bp fragment was found. Although straw length was recognized as an important character already in the 19th century, Rht8 probably played a minor role for height variation. The use of WMS261 and other functional markers for analyses of historical specimens and characterization of historic crop traits is discussed.

Domestication of animals has given rise to a great phenotypic divergence in selected animals and rapidly generated species of animals more accustomed to human contact and social interactions within the species. Previous studies in chickens (Gallus gallus) have managed to find behavioral and adaptive differences between Red Junglefowl (RJF) and White Leghorn (WL), differences inherent to the domestication process. These phenotypic changes could spawn from a variety of different genomic factors, including an epigenetic gene expression regulatory mechanism known as CpG methylation, a DNA modification of CpG dinucleotides that in turn affect nucleosome formation. In this study we investigated the methylation differences between RJF and WL. This by selecting genes that has previously been shown to be both differentially expressed (DE) and differentially methylated (DM) between RJF and WL, and had shown the same kind of differences in both parental animals and their offspring. By using methylation-sensitive high-resolution melting (MSHRM) we tried to confirm previous DM result, and four genes; FUCA1, RUFY3, PCDHAC1 and TXNDC16 were tested and verified to be DM between RJF and WL.

Monitoring presence and level of background DNA in forensic DNA laboratory environments can be used to control work routines and cleaning procedures and to follow changes in these, as well as being an indicator for increased/decreased contamination risk. Previous monitoring routines as sampling and interpretation have not been standardised, making it difficult to compare between different sampling events and observe potential trends. Factor analysis was used to generate a simple graphical classification model for unbiased ranking of electropherograms, which can be modified according to user's need, taking into account number of detected alleles, markers and peak height.

The non-specific lipid transfer proteins (nsLTPs) are small, basic proteins characterized by a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers. Most nsLTPs are synthesized with an N-terminal signal peptide that localizes the protein to the apoplastic space. The nsLTPs have only been identified in seed plants, where they are encoded by large gene families. We have initiated an analysis of the evolutionary history of the nsLTP family using genomic and EST information from non-seed land plants and green algae to determine: (1) when the nsLTP family arose, (2) how often new nsLTP subfamilies have been created, and (3) how subfamilies differ in their patterns of expansion and loss in different plant lineages. In this study, we searched sequence databases and found that genes and transcripts encoding nsLTPs are abundant in liverworts, mosses, and all other investigated land plants, but not present in any algae. The tertiary structures of representative liverwort and moss nsLTPs were further studied with homology modeling. The results indicate that the nsLTP family has evolved after plants conquered land. Only two of the four major subfamilies of nsLTPs found in flowering plants are present in mosses and liverworts. The additional subfamilies have arisen later, during land plant evolution. In this report, we also introduce a modified nsLTP classification system.

Autoimmune Polyendocrine Syndrome Type 1(APS I) is a monogenic autosomal recessive autoimmune disorder which is the result of mutations in the autoimmune regulator (AIRE) gene. Symptoms of the disease include circulation of multiple organ specific autoantibodies, which leads to the breakdown of several tissues, including the adrenal cortex and the parathyroid glands. The patients also develop a number of non-endocrine disorders. This study has investigated the peripheral tolerance mechanisms controlled by the AIRE gene in Aire deficient mice, an animal model of the disease. The B cell Activating Factor (BAFF), which is a cytokine involved in B cell survival and growth, is elevated in Aire^-/- mice, resulting in an increased release of autoantibodies and B cell proliferation. Therefore the BAFF level differences between TCR-/- and B6 mice was studied, and the results showed significantly higher levels of BAFF in TCR^-/- mice. This is not in accordance with earlier studies. ICOS and ICOSL are involved in the activation of follicular T helper cells. The expression of ICOSL on different subpopulations of DC from mice was studied to evaluate the possible influence of AIRE expression on the T cells in the spleen. The results showed that ICOSL is significantly higher expressed in peripheral 33D1+ DCs in Aire^-/- mice, showing that AIRE has a role in the over-activation of the follicular T helper cells, which can lead to autoantibody production and inflammation. These results show that AIRE is involved in peripheral tolerance.

During nervous system development, combinatorial codes of regulators act to specify different neuronal subclasses. However, within any given subclass, there exists a further refinement, apparent in Drosophila and C. elegans at single-cell resolution. The mechanisms that act to specify final and unique neuronal cell fates are still unclear. In the Drosophila embryo, one well-studied motoneuron subclass, the intersegmental motor nerve (ISN), consists of seven unique motoneurons. Specification of the ISN subclass is dependent upon both even-skipped (eve) and the zfh1 zinc-finger homeobox gene. We find that ISN motoneurons also express the GATA transcription factor Grain, and grn mutants display motor axon pathfinding defects. Although these three regulators are expressed by all ISN motoneurons, these genes act in an eve?grn?zfh1 genetic cascade unique to one of the ISN motoneurons, the aCC. Our results demonstrate that the specification of a unique neuron, within a given subclass, can be governed by a unique regulatory cascade of subclass determinants.

The exact origin of the peculiar naked barley is somewhat illusive. There   is a debate whether it has a single, monophyletic origin or a multiple, paraphyletic origin. It is from previous Asian studies on naked   barley known that a mutation   or a deletion of the nud gene expresses the   naked seed phenotype. Not much   investigation has been done outside of   Asia, least of all in the Nordic countries, on what gives naked   barley its character. Therefore this   study was set up to examine if   the Nordic variant of naked barley shares   the same nud allele as the Asian   and thus has a   close connection with it, or   if they have independent mutations. I   could confirm that the known alleles of the nud gene do determine the seed character of barley. Most of the   results of the PCR genotyping confirmed the phenotype of the tested   accessions, both naked and hulled barleys. However, one visually phenotyped naked   barley cultivar (NGB4580) still amplified with the known primers that would   match the Asian hulled allele, meaning that the Nordic accession NGB4580 of   naked barley did not carry the known nud   deletion. This suggests that naked barley has arisen independently in Asia   and in the Nordic countries.

Landrace accessions have long been recognized as an important source of genetic diversity for crop species, and landraces are stored in genebanks across the world as genetic resources for future crop development. Landraces are also an important part of the human cultural heritage and as such they have been used for genetic studies to make inferences about historical agriculture. However, surprisingly little is known about the within-accession diversity of landrace crops of different species. In order to evaluate the diversity of Swedish landraces we used microsatellite markers to genotype accessions of four species (barley, pea, oats and rye), both extant genebank material and 114-year-old seed samples of similar geographic origin and type. We found consistently high levels of within-population genetic diversity in the historical material, but varying and often lower diversity levels in the genebank accessions. We also make tentative conclusions about how representative the genebank material is to what was originally cultivated in its reported area of origin and suggest that the true identity of the genebank accessions is unclear and that historical seed collections should be a more appropriate material for the study of historical agriculture.

In this study we present a comprehensive statistical comparison between a number of reference databases used in Sweden and two databases of DNA profiles from casework. Our results show no substantial differences with respect to various measurements of overall discrepancies but reveal significant differences for individual alleles at several loci.

On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a new analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA® card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog. © 2008 Elsevier Ireland Ltd. All rights reserved.

In 2009–2010, several forensic DNA profiling kits accustomed for Europe and the Prüm Treaty were commerciallyreleased. The manufacturers have made efforts to increase the PCR inhibitor tolerance compared to olderkits, as shown by their increased resistance to known inhibitors such as humic acid and hematin. However, theinhibitor content in true crime scene stains is more complex. Inhibitors may be unknown or not well characterisedand various troublesome compounds may be mixed.Here we evaluate four new 15 STR-marker profiling kits on 29 inhibited crime scene stains from routine caseworkwith DNA concentrations ranging from 0.026 to 0.11 ng/μL. For AmpFℓSTR SGM Plus, used as reference,analysis of 7 samples generated negative/blank DNA profiles, whereas 22 samples produced partial profiles.The four investigated kits were AmpFℓSTR NGM (Applied Biosystems), PowerPlex ESI 16, PowerPlex ESX 16(Promega) and Investigator ESSplex (Qiagen). All four new kits produced DNA profiles of significantly improvedquality compared to AmpFℓSTR SGM Plus. No profiles came out negative/blank. However, the kits were affectedby the complex samples and often failed to produce complete profiles. Matrices such as cigarette butts andmoist snuff seemed especially troublesome. The new kits have raised the bar for PCR inhibitor tolerance, butthe problem still needs to be considered.

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 AmpFlSTR SGM Plus, replacing AmpliTaq Gold DNA polymerase with a customised blend of two alternative polymerases, ExTaq 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.

Crime scene stains often contain extraneous compounds that may interfere with PCR-based DNA analysis,resulting in partial or negative/blank DNA profiles. Extensive DNA purification may remove PCR inhibitors, butinvolve a risk of DNA loss and introduction of contaminations. Customising the chemical content of the PCRreaction is a strategy that may increase PCR inhibitor tolerance without manipulating the sample. Previously wehave shown that crime scene stain analysis can be significantly improved by replacing the commonly used DNApolymerase AmpliTaq Gold with either individual alternative DNA polymerases or a blend of such enzymes [1,2].Here we present the validation of AmpFℓSTR SGM Plus with a modified PCR chemistry for routine casework,applying a 1:1 blend of the DNA polymerases ExTaq Hot Start and PicoMaxx High Fidelity. Allele callings areidentical to standard analysis, and stutters sizes and balance values are indistinguishable. The modified chemistryprovides increased resistance to PCR inhibitors, resulting in an elevated number of detected alleles for crimescene stains of both blood and secretion/saliva. Additionally, the detection limit is improved.[1] Hedman, J., Nordgaard, A., Rasmusson, B., Ansell, R. and Rådström, P. (2009) Improved forensic DNA analysis through the use of alternativeDNA polymerases and statistical modeling of DNA profiles. Biotechniques, 47, 951-958.[2] Hedman, J., Nordgaard, A., Dufva, C., Rasmusson, B., Ansell, R. and Rådström, P. (2010) Synergy between

Background

Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms.

Results

We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles.

Conclusions

The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification.

The Phadebas® Forensic Press test is a new product that detects saliva stains by reacting with amylase. When the paper is pressed against a saliva stain a blue spot occurs. To test the sensitivity of the paper, a set of dilution series of saliva down to 1:500 was prepared on cotton fabric. Blue spots could be seen for dilutions of 1:100 when incubated at room temperature, and 1:200 in 37 °C. However, incubation at 37 °C did not provide acceptable reproducibility. The Phadebas® test was compared to four different lightsources for the ability to detect saliva on different carrier materials. Cotton fabric (T-shirt), denim, suede, leather, painted wood and untreated wood were tested. On denim, no stains could be detected with the lightsources, but Phadebas® showed all stains for pure saliva and dilution 1:5. DNA analysis was performed on detected stains and corresponding spots on the Phadebas® paper. Complete DNA profiles were produced for stains from pure saliva and dilution 1:5.

The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substancespresent in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene salivastains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNApolymerase–buffer systems (Hedman et al., BioTechniques 47 (2009) 951–958). Here we show thatblending inhibitor-resistant DNA polymerase–buffer systems further increases the success rate of PCRfor various types of real crime scene samples showing inhibition. For 34 of 42 ‘‘inhibited” crime scenestains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq HotStart and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmedby analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerasesused separately for all tested sample types. When used separately, the performance of the DNApolymerases varied depending on the nature of the sample. The superiority of the blend is discussed interms of complementary effects and synergy between the DNA polymerase–buffer systems.

DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.

During the water-to-land transition, that occurred approximately 450 MYA, novel habitats wererevealed to the emerging plants. This terrestrial habitat was a harsh environment compared to theaquatic, with shifting substrate content, irregular supply of water, damaging UV-radiation andrapid fluctuating temperatures. Non-specific lipid transfer proteins (nsLTP) are today only foundin the land living plants and not in the green algae. This suggests that these genes might haveevolved to help the plants cope with the stressful conditions. In this study the expression patternhas been analysed of the nsLTPs in the moss Physcomitrella patens during the possible conditionsthat raised during the water-to-land transition. The moss was exposed to salt, UV-B, drought, copper, cold and osmotic stress. Quantitative real-time PCR was used to analyse the transcription levels. I found that six genes were upregulated during either cold, dehydration or UV-B stress. This suggest that these genes are involved in the plant defense against these abiotic stresse

Non-specific lipid transfer proteins is a large and diverse protein family found in plants, with roles in biological systems ranging from long distance signaling to plant pathogen defense. Little is known about the roles of nsLTPs, but recent studies have cast some light on the issue, among other things proposing that they may be involved in the cutice formation on land-living liverworts, mosses and non-seedbearing plants. Increased cuticle formation is thought to be a part of a plants defense system against stress. In this experiment, the expression of nsLTPs type G in the moss Physcomitrella patens was examined by qRT-PCR on cDNA synthesized from already existing mRNA samples from moss under different abiotic stresses. The different stresses were UV-light, salt (ion toxicity), heavy metal, cold drought, plant hormone and osmosis. House-keeping gene P. patens beta-tubuline 1 was used as reference and relative expression analysis was performed. The study showed a general down-regulation of PpLTPg's in the abiotically stressed samples, and the possible coupled regulatory response of PpLTPg3 and PpLTPg5. The results imply that the PpLTPg's in Physcomitrella patens could be connected to biological processes that cease during stress, or that they worl through negative feedback to support plant defense against stress.

Protein-labeling today is a work of art, in vivo studies of proteins or other molecules can easily be performed, and the movement of the labeled molecule can be followed in real time. Labeling in vitro gives enormous amount of data in labs all over the world on a daily basis, where protein-protein, protein-DNA or other interactions are studied. Folding and unfolding events can be monitored wi th labels reporting on local or global environmental changes in a protein. The use of labeling seems endless, but in this thesis I have chosen to focus on two labeling techniques: spin-labeling and fluorescence labeling. Applying these techniques on protein-protein and protein-DNA interactions has resulted in better understanding of protein folding and function.

Chaperonin function at elevated temperatures

The model protein HCA II (259 amino acids) mainly consisting of a large 10 stranded ß-sheet with a topological breakpoint between strands 6 and 7. Two residues, adjacent in the folded structure and located at each side of this breakpoint, were used in a site-directed-spin-labeling (SDSL) experiment. The aim was to elucidate what happens at the breakpoint when the protein interacts with the chaperonin GroEL at elevated temperatures. The chaperonin GroEL is a 60 kDa protein known to aid protein folding in the cell. By probing the model protein, HCA II, we have shown that this chaperone can stretch its substrate and release it for a new refolding opportunity.

MexR protein interaction with DNA

MexR is a 147 amino acid protein dimer involved in transcriptional repression of the multidrug efflux operon MexAB-OprM in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Malfunction in MexR results in multidrug resistant bacteria resistant to therapeutic strategies. Site-specific fluorescence-labeling of MexR has been investigated as a means to provide a new strategy for localising DNA binding and quantifyi ng DNA affinity. ANS fluorescence of the MexR protein in the absence and presence of DNA, together with a range of biophysical measurements, has provided a new view on how MexR could be regulated by small molecule binding, and thus sheds new light on its functionality in gene repression.

Plant non specific lipid transfer proteins (nsLTPs) enhance in vitro transfer of phospholipids between membranes. Our analysis exploited the large amount of Arabidopsis transcriptome data in public databases to learn more about the function of nsLTPs. The analysis revealed that some nsLTPs are expressed only in roots, some are seed specific, and others are specific for tissues above ground whereas certain nsLTPs show a more general expression pattern. Only few nsLTPs showed a strong up or downregulation after that the Arabidopsis plant had suffered from biotic or abiotic stresses. However, salt, high osmosis and UV-B radiation caused upregulation of some nsLTP genes. Further, when the coexpression pattern of the A.thaliana nsLTPs were investigated, we found that there were several modules of nsLTP genes that showed strong coexpression indicating an involvement in related biological processes. Our finding reveals that the nsLTPs gene was significantly correlated with lipase and peroxidase activity. Hence we concluded that the nsLTPs may play a role in seed germination, signalling and ligning biosynthesis.

This study was initiated to investigate a possible strategy to alter an enzyme deficiency in a mouse model. The enzyme investigated is a multifunctional nucleoside kinase from Drosophila melanogaster (Dm-dNK). This enzyme has special features in that it has higher enzymatic activity than any other known nucleoside kinases and still has similar substrate specificity as the human nucleoside kinases. The deficiency where the Dm-dNK transgenic mice model will be used is a TK2 deficient model with severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK transgenic mice model will be used as a way to rescue the TK2 deficient mice. The results from the present study show that Dm-dNK expression in mice results in a substantial increase of thymidine phosphorylation in several investigated tissues. The mice were otherwise normal as judged by life span, weight and behavior. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK mouse model is promising as a way to rescue the severe phenotype of the TK2 deficient mice.