29 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 6â8 hours
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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
Complex probes for high-throughput parallel genetic mapping of genomic mouse BAC clones
We describe a novel approach for the identification and mapping of polymorphic markers. Amplicons are generated by ligation of double-stranded adaptor molecules to genomic DNA cleaved with a restriction enzyme. Using primers that extend beyond the restriction site, reduced-complexity subsets of fragments are generated by PCR. Differences in the composition of complex probes generated from DNA of different strains are revealed through hybridization against high-density filter grids of large-insert genomic clones. Genetic mapping of genomic clones is achieved by hybridizing complex probes derived from backcross animals against the polymorphic clones. The mouse was chosen as a model system to test the feasibility of this technique because of the general availability of backcross resources and genomic libraries. Nevertheless, we would expect the method to be of particular use to generate markers for species that have not yet been extensively studied, because a substantial number of easy-to-use markers can be recruited in a relatively short period of time
Genome Res.
We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing 10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual
Contraintes et fouille de données
La fouille de donnĂ©es est un domaine de recherche actif, visant Ă dĂ©couvrir des connaissances implicites dans des bases de donnĂ©es. Nous Ă©tudions ici l'intĂ©rĂȘt de formalismes issus de la logique du premier ordre pour la fouille de donnĂ©es. En particulier, nous examinons l'intĂ©rĂȘt des contraintes, vues comme des formules du premier ordre et interprĂ©tĂ©es sur un domaine particulier. Un point important de tout formalisme utilisĂ© en ECD est la dĂ©finition d'une relation de gĂ©nĂ©ralitĂ© qui permet de structurer l'espace des motifs, et de faciliter ainsi la recherche de motifs intĂ©ressants. Nous nous intĂ©ressons tout d'abord aux bases de donnĂ©es contraintes qui Ă©tendent Ă la fois les bases de donnĂ©es relationnelles, dĂ©ductives et spatiales, et qui permettent la dĂ©finition d'ensembles infinis grĂące Ă la notion de tuples gĂ©nĂ©ralisĂ©s. Nous Ă©tudions ensuite le formalisme des clauses contraintes utilisĂ©es en Programmation Logique avec Contraintes. Nous reprenons la dĂ©finition classique de gĂ©nĂ©ralitĂ© entre clauses contraintes et nous dĂ©terminons dans ce cadre le moindre gĂ©nĂ©ralisĂ©, le moindre spĂ©cialisĂ© et des opĂ©rateurs de raffinement. Nous montrons comment les calculer en pratique sur plusieurs domaines de contraintes spĂ©cifiques. Enfin nous introduisons un nouveau motif: les rĂšgles caractĂ©ristiques. Ces rĂšgles sont la combinaison d'un chemin quantifiĂ© et d'une contrainte et permettent de capturer la notion de lien entre entitĂ©s et de contraintes sur ces entitĂ©s. Nous montrons l'intĂ©rĂȘt de telles rĂšgles dans le cadre de bases de donnĂ©es gĂ©ographiques, notamment sur des donnĂ©es gĂ©ologiques fournies par le Bureau de Recherche GĂ©ologique et MiniĂšres.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF
Blood Cells Mol. Dis.
To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing - and -globin genes from the pufferfish Fugu rubripes. By DNA FISH analysis we show that Fugu contains two distinct hemoglobin loci situated on separate chromosomes. One locus contains only -globin genes ( -locus) while the other also contains a -globin gene ( -locus). This is the first poikilothermic species analysed where the physical linkage of the - and -globin genes has been uncoupled, supporting a model in which the separation of the - and -globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu -locus suggest that a strong distal Locus Control Region regulating the activity of the globin genes, as found in mammalian -globin clusters, may not be present in the Fugu -locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are in close proximity to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the five human globin loci known to date, each encoding proteins with specific functions in specific cell types
The Organellar Genome and Metabolic Potential of the Hydrogen-Producing Mitochondrion of Nyctotherus ovalis
It is generally accepted that hydrogenosomes (hydrogen-producing organelles) evolved from a mitochondrial ancestor. However, until recently, only indirect evidence for this hypothesis was available. Here, we present the almost complete genome of the hydrogen-producing mitochondrion of the anaerobic ciliate Nyctotherus ovalis and show that, except for the notable absence of genes encoding electron transport chain components of Complexes III, IV, and V, it has a gene content similar to the mitochondrial genomes of aerobic ciliates. Analysis of the genome of the hydrogen-producing mitochondrion, in combination with that of more than 9,000 genomic DNA and cDNA sequences, allows a preliminary reconstruction of the organellar metabolism. The sequence data indicate that N. ovalis possesses hydrogen-producing mitochondria that have a truncated, two step (Complex I and II) electron transport chain that uses fumarate as electron acceptor. In addition, components of an extensive protein network for the metabolism of amino acids, defense against oxidative stress, mitochondrial protein synthesis, mitochondrial protein import and processing, and transport of metabolites across the mitochondrial membrane were identified. Genes for MPV17 and ACN9, two hypothetical proteins linked to mitochondrial disease in humans, were also found. The inferred metabolism is remarkably similar to the organellar metabolism of the phylogenetically distant anaerobic Stramenopile Blastocystis. Notably, the Blastocystis organelle and that of the related flagellate Proteromonas lacertae also lack genes encoding components of Complexes III, IV, and V. Thus, our data show that the hydrogenosomes of N. ovalis are highly specialized hydrogen-producing mitochondria