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de Sequenciamento e Análise de Microssatélites.
Artigo que descreve as primeiras análises dos resultados do Projeto genoma Humano
Abstract: The human genome holds an extraordinary trove of information about human development, hysiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
Um resumo e perspectivas das principais técnicas de sequenciamento de DNA, incluindo as novas plataformas de sequenciamento em larga escala
Abstract: The decade since the Human Genome Project ended has witnessed a remarkable sequencing technology explosion that has permitted a multitude of questions about the genome to be asked and answered, at unprecedented speed and resolution. Here I present examples of how the resulting information has both enhanced our knowledge and expanded the impact of the genome on biomedical research. New sequencing technologies have also introduced exciting new areas of biological endeavour. The continuing upward trajectory of sequencing technology development is enabling clinical applications that are aimed at improving medical diagnosis and treatment.
Poster que demonstra a possibilidade de sequenciamento de DNA diretamente a partir de células em cultura, sem extração e PCR
Abstract: Genome centers are continually working to increase the throughput and decrease the reagent costs and manual labor of their sequencing process, including the plasmid template preparation step. Researchers have attempted to bypass this step by directly sequencing from bacterial cultures, but in the past the results have been considerably inferior to standard purified plasmid sequencing, with lower success rate and shorter read lengths. Using BigDye® Terminator chemistry on the Applied Biosystems 3730xl DNA Analyzer, we have recently obtained very promising results—we are able to produce read lengths and success rates comparable to high throughput purified plasmid sequencing. Here we will present the results of our studies to optimize direct culture sequencing from various template types, using 96 and 384-well format. We have also seen promising results with direct sequencing from colonies. This allows much faster screening of libraries for sequences of interest.
Abstract: Bacterial artificial chromosome (BAC) DNA is difficult to use as a sequencing template, because of its large size (usually >100,000 bp). A modified sequencing protocol has been demonstrated for BAC DNA using the BigDye® Terminator Cycle Sequencing Kit. Additionally, implementation of the BigDye® XTerminator™ Purification Kit for reaction cleanup resulted in increased signal intensity and overall reading quality of the sequence data comparable to that from plasmid templates.
Abstract: Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost-effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next-generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non-model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use ofmultiplex PCR. To make the most of the latest technical developments, we outline the need for a well-established strategy including standardized high-throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.
Artigo que descreve uma metodologia alternativa para triagem de locos informativos de microssatélites para estudos populacionais.
Abstract: Drescribes the procedure for fluorescent dye labeling of PCR fragments in one reaction, which is performed with three primers: a sequence-specific forward primer with M13(-21) tail at its 5' end, a sequence-specific reverse primer, and the universal fluorescent-labeled M13(-21) primer.
Abstract: Free fluorescent dyes from PCR primers or amplification products can interfere with the interpretation of STR alleles in an electropherograph especially when the profiles have a low signal intensity. These artefacts can be removed by using a simple procedure based on BigDye1 XTerminatorTM. This procedure requires limited amounts of PCR product, allows to do several loadings on a capillary sequencer starting from the same purified PCR product and also increases the sensitivity for detection of less amplified loci.
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