<p>Det har nyligen skett ett mord i salladsdisken i närbutiken. Så börjar inledningen till detta arbete. Syftet med studien var att ta fram en PCR-baserad genetiklaboration med RAPD-primer som är lärorik och intressant för gymnasielever i de naturvetenskapliga ämnena. RAPD är en metod där endast en primer används, till skillnad från de flesta andra PCR-metoder som använder två stycken primer. Laborationen har aldrig gjorts förut, vilket lett till att en stor del av arbetets tyngd ligger i att metodutveckla laborationen och sedan testa laborationen på en testgrupp. Testgruppen bestod av 9 gymnasieelever som läser naturkunskap B. Resultatet visade att 8 av de 9 elever som gjorde laborationen ansåg att laborationen bör ingå i naturkunskap B. Enkätundersökningen visar även att genetiklaborationen med RAPD primer höjde intresset för genetik och genteknik hos försökspersonerna. Slutsatsen är att laborationen lämpar sig mycket väl för att öka intresset för genetik och genteknik bland gymnasieelever.</p>

<p>Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis.</p><p>Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus.</p><p>Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined.</p><p>Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested.</p><p>Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.</p>

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFRα-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFRα-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFRα-2b and GFRα-2c). The expression levels of these isoforms have yet to be quantified and the functional properties determined. In this report, we have developed a real time polymerase chain reaction (PCR) using SYBR Green I to detect the expression levels of the three splice variants (GFRα-2a, GFRα-2b and GFRα-2c) in murine tissues. Both GFRα-2a and GFRα-2c were expressed at similar levels in all tissues examined. GFRα-2b was found to be 10 fold lower in expression. All three isoforms activated MAPK (ERK1/2) and Akt. Transcriptional profiling with DNA microarrays demonstrated that the spliced isoforms do not share similar profiles. In conclusion, we have now shown the expression levels of the spliced variants. All three isoforms are functional. However, each isoform appeared to have unique transcriptional profiles when activated. / Singapore-MIT Alliance (SMA)

Steroid hormones, such as estrogen, are produced in one tissue and carried through the blood stream to target tissues in which they bind to highly specific nuclear receptors and trigger changes in gene expression and metabolism. Industrial chemicals, such as bisphenol A and many agricultural chemicals, including permethrin and fervalerate, are known to have estrogenic potential and therefore are estrogen mimics. Widely used agricultural chemicals, Enable (fungicide) and Diazinon (insecticide), were evaluated to examine their toxicity and estrogenicity. MCF-7 cells, an estrogen-dependent human breast cancer line, were utilized for this purpose. MCF-7 cells were treated with 0.033-3.3 ppb (ng/ml) of Enable and 0.3-67 ppm of Diazinon and gene expression was compared to that in untreated cells. Microarray analysis showed down-regulation of eight genes and up-regulation of thirty four genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Similarly, in cells treated with 67 ppm of Diazinon, there were three genes down-regulated and twenty seven genes up-regulated. For both chemicals, specific genes were selected for special consideration. RT-PCR confirmed results obtained from analysis of the microarray. These studies were designed to provide base-line data on gene expression-altering capacity of specific chemicals and will allow assessment of the deleterious effects caused by exposure to the aforementioned chemicals.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.

Bei konventionellen Mikroarray-Experimenten zur Genexpressionsanalyse wird fluoreszenz- oder radioaktiv-markierte cDNA oder RNA mit immobilisierten Proben hybridisiert. Für ein gut detektierbares und auswertbares Ergebnis werden jedoch pro Array mindestens 15 - 20 &#181;g Hybridisierungstarget benötigt. Dazu müssen entweder 15 - 20 &#181;g RNA direkt durch Reverse Transkription in markierte cDNA umgeschrieben werden oder bei Vorhandensein von weniger Startmaterial die RNA amplifiziert werden (Standard- Affymetrix-Protokolle, Klur et al. 2004). Oft sind damit zeit- und kostenintensive Probenpräparationen verbunden und das Ergebnis ist nicht immer reproduzierbar. Obwohl es inzwischen einige Protokolle gibt, die dieses Problem zu lösen versuchen (Zhang et al. 2001, Iscove et al. 2002, McClintick et al. 2003, Stirewalt et al. 2004), eine optimale, leicht handbare und reproduzierbare Methode gibt es weiterhin nicht, weshalb in dieser Arbeit ein weiterer Lösungsansatz gesucht wurde.<br> In der vorgestellten Arbeit werden zwei einfache Methoden beschrieben, mit denen Gene aus geringen RNA-Mengen nachgewiesen werden können: erstens die On Chip- RT-PCR mit cDNA als Matrize und zweitens diese Methode als One-Step-Reaktion mit RNA als Matrize.<br><br> Beide Methoden beruhen auf dem Prinzip der PCR an immobilisierten Primern auf einer Chipoberfläche. Diese Möglichkeit der exponentiellen Amplifikation ist reproduzierbar und sensitiv.<br><br> In Experimenten zur Etablierung des On-Chip-PCR-Systems wurden für die Immobilisierung der Primer verschiedene Kopplungsmethoden verwendet. Die affine Kopplung über Biotin- Streptavidin erwies sich als geeignet. Die On-Chip-Reaktion an kovalent gebundenen Primern wurde für amino-modifizierte Primer auf Epoxy-Oberflächen sowie für EDC-Kopplung auf silanisierten Oberflächen gezeigt. Für die letztgenannte Methode wurde die On-Chip-PCR optimiert, dass Spottingkonzentrationen der Primer von 5 - 10&#181;M schon ausreichend sind. Der Einsatz von fluoreszenz-markierten Primern während der PCR ermöglicht eine unmittelbare Auswertung nach der Synthese ohne zusätzliche Detektionsschritte. In dieser Arbeit konnte außerdem mit der vorgestellten Methode der simultane Nachweis zweier Gene gezeigt werden. Die Methode kann noch als Multiplex-Analyse ausgebaut werden, um so mehrere Gene in gleichzeitig einem Ansatz nachweisen zu können.<br><br> Die Ergebnisse der Versuche mit Matrizen aus unterschiedlichen Zelltypen deuten darauf hin, dass die On-Chip-RT-PCR eine weitere optimale Methode für den Nachweis von gering exprimierten Genen bietet. / The detection of low quantities of mRNA is often difficult and methods like microarray analysis require large amount of starting material or have to be amplified before application. The reverse transcription polymerase chain reaction (RT-PCR) is often the chosen method to detect specific RNA sequences on account of its high sensitivity. The solid phase amplification technology by On-Chip-PCR provides a combination of amplification of rare target material and its on chip detection in one step.<br><br> In this report a novel application for the On-Chip-PCR technology is described. It was suitable to identify mRNA sequences and genes, respectively. For this approach we amplified cDNA sequences using immobilized specific primers and fluorescent labeled primers. They were used for genes coding subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm) and Atpase (Atp2a2). The cDNA templates were synthesized before the On-Chip application by Reverse Transcription from mRNA. For this application only at most 500 pg of total-RNA preparation was sufficient for detectable results and no pre-amplification steps were needed.<br><br> Furthermore the handling of RT-PCR could be minimized by using a One-step- RT-PCR protocol. This method used immobilized primers on glassy supports detecting specific mRNA sequences from 5 pg or less of total RNA preparations.<br> Moreover to the detection of low quantities of RNA preparation, low abundant genes could be detected by this method.

Fusobacterium necrophorum är en gramnegativ anaerob bakterie som grupperas i ss. necrophorum och ss. funduliforme. Fusobacterium necrophorum ss. funduliforme har på senare tid misstänkts kunna spela en roll vid vanligare svalginfektioner såsom halsfluss. Syftet med detta arbete var att sätta upp och bepröva en metod för realtids-PCR (Polymerase Chain Reaction) enligt Taqman för att detektera ss funduliforme. Vi undersökte även hur förvaringstiden i transportmedium (Amies kol, Copan) och odlingsmedium (Fastidious Agar Broth) påverkar överlevnaden för F. necrophorum ss. funduliforme och resultatet från realtids-PCR. Bakteriesuspension av varierande koncentration applicerades på provtagningspinnar som direkt inkuberades i medium, varefter en pinne av vardera koncentration utodlades och DNA extraherades för varje dag försöket pågick. En serie 10-spädningar av extraherat DNA från ss funduliforme ,med en lägsta koncentration av 10 DNA-molekyler per µL, användes som standard och positiv kontroll för PCR. Fusobacterium necrophorum ss funduliforme kunde detekteras med realtids-PCR från alla prov under alla dagar försöket pågick, medan odlingarna visade bäst resultat om provet såddes ut inom ett dygn från provtagningen. Realtids-PCR kan därför detektera förekomsten av ss funduliforme efter längre förvaring och bör användas för exempelvis transporterade prover, medan traditionell utodling med fördel kan användas för diagnostering av ss fundulforme då PCR inte ger information om antibiotikaresistens hos isolatet. / Fusobacterium necrophorum are Gram-stain negative, anaerobic, bacteria that are grouped into subspecies necrophorum and funduliforme. Fusobacterium necrophorum ss. funduliforme has recently been suspected to play a role in common throat infections such as tonsillitis. The purpose of this work was to set up and test a method for real-time PCR (Polymerase Chain Reaction) according to Taqman with the purpose of detecting ss. funduliforme. We also examined how the storage time within transport medium (Amies charcoal, Copan) and culture medium (FAB-broth) affects the survival of the bacterium and the sensitivity of the culture and PCR methods. Bacterial suspensions of different concentrations were applied to pharyngeal sampling swabs and then directly incubated in transport medium. For each day the experiment lasted, a set of swabs of each concentration were cultured, DNA extracted, and real-time PCR performed. DNA extracted from ss. funduliforme was used as standards for the real-time PCR, with a minimum concentration of 10 DNA molecules per μL. Subspecies funduliforme could be detected from all days with real-time PCR while the cultures showed the best results if the sample was cultured within one day of collection. Real-time PCR can detect the presence of ss. funduliforme after prolonged storage and can therefore show accurate results for transported samples. Traditional culturing on growth medium is still a valuable and reliable method, provided that the samples are cultured within 24 hours. Culture may also be needed i.e. since PCR gives no information of the antibiotic resistance of the isolate.

Nitrification is a natural occurring, oxidative process which is essential for plants´ ability to take up nitrogen in the form of nitrate. The oxidation is divided into two steps. First ammonia is oxidized to nitrite by ammonia-oxidizing bacteria (AOB) or archaea (AOA) and then the nitrite is further oxidized to nitrate by nitrite-oxidizing bacteria (NOB). The enzyme used by NOB for the oxidation is nitrite oxidoreductase (nxr). One of few bacteria that catalyze this reaction is Nitrobacter sp. The purpose of this study has been to optimize the detection of Nitrobacter in samples of activated sludge from municipal wastewater treatment plants (WWTPs) in Eskilstuna and Västerås (Sweden). This was done by PCR, cloning and sequencing. Primers used were nor F/nor R that are specific for the functional gene encoding nxr. This optimization has been compared to a different PCR-system where nor F/nor R were exchanged for another primerpair consisting of a 16S rDNA-primer (NIT3), which was specific for Nitrobacter and a universal 16S-primer (U2, Rit388). In addition to this, a semi quantitative analyze has also been conducted. The result of the study was two PCR-programmes, one optimized for each set of sludge samples. The quantitative analysis showed that the concentration Nitrobacter in the sludge samples was approximately the same as a pure culture, which was used as a positive control and contained ~104 CFU/ml. Cloning and sequencing revealed the presence of 3 different Nitrobacter. Surprisingly half of the clones from one of the Västerås samples, taken in December, were most likely Methylibium petroleiphilum. The matter of fact that we were able to detect this bacterium with primers specifically designed for Nitrobacter made this discovery even more interesting. With NIT3/U2 Methylocella sp. was also detected in samples from Västerås, which confirmed the presence of methylotrophic bacteria in the Västerås samples. / Activated sludge process optimization

Det har nyligen skett ett mord i salladsdisken i närbutiken. Så börjar inledningen till detta arbete. Syftet med studien var att ta fram en PCR-baserad genetiklaboration med RAPD-primer som är lärorik och intressant för gymnasielever i de naturvetenskapliga ämnena. RAPD är en metod där endast en primer används, till skillnad från de flesta andra PCR-metoder som använder två stycken primer. Laborationen har aldrig gjorts förut, vilket lett till att en stor del av arbetets tyngd ligger i att metodutveckla laborationen och sedan testa laborationen på en testgrupp. Testgruppen bestod av 9 gymnasieelever som läser naturkunskap B. Resultatet visade att 8 av de 9 elever som gjorde laborationen ansåg att laborationen bör ingå i naturkunskap B. Enkätundersökningen visar även att genetiklaborationen med RAPD primer höjde intresset för genetik och genteknik hos försökspersonerna. Slutsatsen är att laborationen lämpar sig mycket väl för att öka intresset för genetik och genteknik bland gymnasieelever.

In this study the composition of the denitrifying bacterial community among constructed wetlands in agricultural areas was investigated. Thirty-two constructed wetlands located in Southern Sweden were surveyed, and biofilm samples from each were analyzed by applying denaturing gradient gel electrophoresis, to investigate the community composition of the three denitrifying bacterial enzyme genes nirK, nirS and nosZ. The DNA sequences of the enzyme genes were compared to known DNA sequences in GeneBank using BLAST. The results of the denitrifying bacterial enzyme genes indicated that these habitats may harbour a heterogeneous denitrifying bacterial community. Individual analysis of the enzyme genes revealed that nirS was more heterogeneous than both nirK and nosZ. Most sequences from the present study clustered with known sequences from species belonging to the group of α-Proteobacteria, and to a lesser extent with β- Proteobacteria and γ-Proteobacteria, and only nirS clustered with a member of gram-positive bacteria. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>