19 research outputs found
Single-nucleotide polymorphisms: analysis by mass spectrometry
Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6–8 hours
SNP-specific extraction of haplotype-resolved targeted genomic regions
The availability of genotyping platforms for comprehensive genetic analysis of complex traits has resulted in a plethora of studies reporting the association of specific single-nucleotide polymorphisms (SNPs) with common diseases or drug responses. However, detailed genetic analysis of these associated regions that would correlate particular polymorphisms to phenotypes has lagged. This is primarily due to the lack of technologies that provide additional sequence information about genomic regions surrounding specific SNPs, preferably in haploid form. Enrichment methods for resequencing should have the specificity to provide DNA linked to SNPs of interest with sufficient quality to be used in a cost-effective and high-throughput manner. We describe a simple, automated method of targeting specific sequences of genomic DNA that can directly be used in downstream applications. The method isolates haploid chromosomal regions flanking targeted SNPs by hybridizing and enzymatically elongating oligonucleotides with biotinylated nucleotides based on their selective binding to unique sequence elements that differentiate one allele from any other differing sequence. The targeted genomic region is captured by streptavidin-coated magnetic particles and analyzed by standard genotyping, sequencing or microarray analysis. We applied this technology to determine contiguous molecular haplotypes across a ∼150 kb genomic region of the major histocompatibility complex
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The archaeology of the military orders: the material culture of holy war
This paper reviews the current state of research into the archaeology of the military orders. It contrasts the advances made by historians and archaeologists, with the latter continuing to focus on the particularism of individual sites, with an emphasis on architectural analyses. Historians have contributed new insights by adopting a supranational approach. This paper argues that archaeologists can build on this by adopting a more problem-oriented, comparative approach. Drawing on examples from frontier and heartland territories, archaeological approaches are subdivided into material investment, material identity and cultural landscapes, to place sites of the military orders within a long-term, multi-scalar contexts. This contributes to a broader social and economic understanding of the orders, who contributed significantly to urbanisation, rural development and trade, and invested in material expressions of their authority and ideology. The paper concludes that more holistic, inter-regional approaches will move the archaeological study of the military orders forward
Generation of two spotted plasmid cDNA-libraries of the head region of stage 20 and whole stage 17 Medaka embryos.
Generation of two spotted plasmid cDNA-libraries of the head region of stage 20 and whole stage 17 Medaka embryos.
Contraintes et fouille de données
La fouille de données est un domaine de recherche actif, visant à découvrir des connaissances implicites dans des bases de données. Nous étudions ici l'intérêt de formalismes issus de la logique du premier ordre pour la fouille de données. En particulier, nous examinons l'intérêt des contraintes, vues comme des formules du premier ordre et interprétées sur un domaine particulier. Un point important de tout formalisme utilisé en ECD est la définition d'une relation de généralité qui permet de structurer l'espace des motifs, et de faciliter ainsi la recherche de motifs intéressants. Nous nous intéressons tout d'abord aux bases de données contraintes qui étendent à la fois les bases de données relationnelles, déductives et spatiales, et qui permettent la définition d'ensembles infinis grâce à la notion de tuples généralisés. Nous étudions ensuite le formalisme des clauses contraintes utilisées en Programmation Logique avec Contraintes. Nous reprenons la définition classique de généralité entre clauses contraintes et nous déterminons dans ce cadre le moindre généralisé, le moindre spécialisé et des opérateurs de raffinement. Nous montrons comment les calculer en pratique sur plusieurs domaines de contraintes spécifiques. Enfin nous introduisons un nouveau motif: les règles caractéristiques. Ces règles sont la combinaison d'un chemin quantifié et d'une contrainte et permettent de capturer la notion de lien entre entités et de contraintes sur ces entités. Nous montrons l'intérêt de telles règles dans le cadre de bases de données géographiques, notamment sur des données géologiques fournies par le Bureau de Recherche Géologique et Minières.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF
A Fosmid Pool-Based Next Generation Sequencing Approach to Haplotype-Resolve Whole Genomes
Haplotype resolution of human genomes is essential to describe and interpret genetic variation and its impact on biology and disease. Our approach to haplotyping relies on converting genomic DNA into a fosmid library, which represents the entire diploid genome as a collection of haploid DNA clones of ~40 kb in size. These can be partitioned into pools such that the probability that the same pool contains both parental haplotypes is reduced to ~1 %. This is the key principle of this method, allowing entire pools of fosmids to be massively parallel sequenced, yielding haploid sequence output. Here, we present a detailed protocol for fosmid pool-based next generation sequencing to haplotype-resolve whole genomes including the following steps: (1) generation of high molecular weight DNA fragments of ~40 kb in size from genomic DNA; (2) fosmid cloning and partitioning into 96-well plates; (3) barcoded sequencing library preparation from fosmid pools for next generation sequencing; and (4) computational analysis of fosmid sequences and assembly into contiguous haploid sequences.This method can be used in combination with, but also without, whole genome shotgun sequencing to extensively resolve heterozygous SNPs and structural variants within genomic regions, resulting in haploid contigs of several hundred kb up to several Mb. This method has a broad range of applications including population and ancestry genetics, the clinical interpretation of mutations in personal genomes, the analysis of cancer genomes and highly complex disease gene regions such as MHC. Moreover, haplotype-resolved genome sequencing allows description and interpretation of the diploid nature of genome biology, for example through the analysis of haploid gene forms and allele-specific phenomena. Application of this method has enabled the production of most of the molecular haplotype-resolved genomes reported to date