Made available in DSpace on 2015-03-26T13:42:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 897621 bytes, checksum: e2a579de88f1b70bef922be819992a6c (MD5) Previous issue date: 2013-03-19 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The fluorescente in situ hybridization advent introducted the cytogenetics in molecular era, with methodological advances that enable the investigation of chromosome science at different levels. Among these advances, it stands out the unequivocally identification of homologous chromosomes pairs, the specific sequences location, the direct prospecting of chromosomal abnormalities, besides providing high-resolution information on the structure and organization of chromosomes. Within the strategies of molecular cytogenetics, microdissection and DOP-PCR techniques have been widely used in the chromosome-specific probes construction. However, the absence of well-established and reproducible protocols, besides the need for relatively large number of chromosomes for micromanipulation, this technique has been limited. The present study aimed to standard a methodology for chromosome-specific probes construction, involving micromanipulation and DOP-PCR techniques. Human lymphocyte cultures were carried out to obtain metaphases, both male and female origin. In situ amplification (PRINS) reaction was performed on a slide containing metaphase, applying 50 μL of reaction mix with Platinun® Taq DNA Polymerase High Fidelity 1 U, enzime reaction buffer, MgSO4 2 mM, dNTPs 200 μM and primer 4 μM. Then, ten X chromosomes fragments were microdissected using microneedles coupled to a phase contrast microscope and transferred to a tube containing 0.2 μL proteinase K, where deproteinizated at 37 °C for 24 hours. The material was amplified by DOP-PCR in 15 μL of reaction, and marked with Tetramethyl-rhodamine-5-dUTP. The probe was denatured for 10 minutes at 99 °C in 35 μL of hybridization mix containing: formamide 50 %, SSC 2X, Dextran Sulfate 10 %, 1 mg of Cot-1 and 200 ng of labeled probe. This mix was applied on the slide, which was denatured at 75 ºC for 8 minutes and then subjected to hybridization at 37 °C for 20 hours. After hybridization, stringency washes and counter-staining with DAPI were performed, and the material was analyzed with a fluorescence microscope coupled to an image capture system. A subcentromeric X chromosome specific probe was obtained, with an unique mark in male metaphase and two marks in female metaphases, with one or two fluorescent signals in interphase nuclei. / Com o advento da hibridização in situ fluorescente, a citogenética foi introduzida na era molecular, com avanços metodológicos que possibilitaram a investigação em vários níveis da ciência dos cromossomos. Destaca-se entre esses avanços a identificação de pares de cromossomos homólogos de forma inequívoca, a localização de sequências específicas, a prospecção direta de anormalidades cromossômicas, além de proporcionar informações de alta-resolução sobre a estrutura e organização dos cromossomos. Dentro das estratégias da citogenética molecular, as técnicas de microdissecção e de DOP-PCR têm sido muito utilizadas na construção de sondas cromossomo-específicas. Entretanto, a ausência de protocolos bem estabelecidos e reprodutíveis, além da necessidade de captura por micromanipulação de um número relativamente grande de cromossomos, essa técnica tem sido limitada quanto à sua aplicação mais ampla. O presente trabalho teve como objetivo padronizar uma metodologia para construção de sondas cromossomo-específicas, associando as técnicas de micromanipulação e DOP-PCR. Culturas de linfócitos humanos foram realizadas para a obtenção de lâminas contendo metáfases, tanto de origem masculina quanto feminina. A reação de amplificação in situ (PRINS) foi realizada sobre uma lâmina contendo metáfases, aplicando uma mistura de 50 μl de reação, contendo 1,0 U de Platinun® Taq DNA Polymerase High Fidelity, tampão de reação da enzima, 2 mM de MgSO4, 200 μM de dNTPs e 4 μM de primer. Em seguida, dez fragmentos de cromossomos X foram microdissectados utilizando microagulhas acopladas a um microscópio de contraste de fase e transferidos para um tubo de 0,2mL contendo proteinase K, de onde seguiu para a desproteinização a 37 ºC por 24 horas. O material foi amplificado por DOP-PCR em 15 μl de reação, e marcado com Tetramethyl-rhodamine-5-dUTP. A sonda foi desnaturada por 10 minutos a 99 ºC em 35 μL de uma mistura de hibridização contendo: 50 % Formamida, 2X SSC, 10 % Dextran Sulfato, 1 μg de Cot-1 e 200 ng da sonda marcada. Essa mistura foi aplicada na lâmina, que foi desnatura a 75 ºC por 8 minutos e depois foi submetida à hibridização a 37 ºC por 20 horas. Após a hibridização, foram realizadas as lavagens de estringência, contra-coloração com DAPI, e visualização do material em microscópio de fluorescência acoplado a um sistema de captura de imagens. Uma sonda específica para a região subcentromérica do cromossomo X foi obtida, apresentando uma marcação em metáfases de indivíduos masculinos e duas marcações em metáfases de indivíduos femininos, além de um ou dois sinais fluorescentes nos núcleos interfásicos.

When forensic biological samples yield low quality/low quantity DNA, thecurrent STR analysis methods do not generate acceptable profiles. Whole genomeamplification can be used to pre-amplify the entire genome for downstream analyses. A commercially available kit for DOP-PCR, a form of WGA, is currently being used in the clinical for downstream single locus targets. Forensic analyses utilize a multiplex amplification. This study determined that the "home brew" created by our lab performs the same as the commercially available kit. Future optimization studies of DOP-PCR can utilize this "home brew". Additionally, this research determined that a 10 second increase in electrokinetic injection time onto the Capillary Electrophoresis (CE) in combination with a post-STR amplification purification and elution into formamide produces a slightly higher percent STR allele success over the standard protocol. After future optimization studies, this may be a useful method to obtain more accurate and complete STR profiles from low quality/low quantity biological samples.

Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen. / Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.

Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification of damaged DNA from both contemporary and historical skeletal remains compared with that obtained by standard DNA typing and a previously described DOP-PCR method. These new DOP-PCR primers show promise for WGA of degraded DNA.