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BAC Library Construction

DNA cloning, especially large DNA cloning, is the first step in contemporary complex genome analysis. Cloning technology of high-molecular-weight DNA has been developed mainly using yeast and Escherichia coli as hosts. In the early stages of the Human Genome Project, yeast artificial ch ...

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Using the TIGR Assembler in Shotgun Sequencing Projects

The TIGR Assembler (TA) (1) is the sequence assembly program used in sequencing projects at The Institute for Genomic Research (TIGR). Development of the TA was based on the experience obtained in more than 20 sequencing projects completed at TIGR (see http://www.tigr.org). This extensive exp ...

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Finishing Working Draft BAC Projects by Directed Sequencing With ThermoFidelase and Fimers

A typical “working draft” bacterial artificial chromosome (BAC) project consists of approx 2000 shotgun reads obtained from a library of M13 or plasmid subclones that provide 2–5X coverage of BAC sequence. The reads are assembled into 5–50 long contigs (2 kb), and hundreds of smaller contigs and ...

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Optimized Multiplex Polymerase Chain Reaction: An Effective Method for Rapid Gap Closure

Implementation of the whole-genome shotgun sequencing approach to prokaryotes and eukaryotes necessitates methods for rapid gap closure. During a whole-genome shotgun sequencing project, DNA sequences are aligned against each other to create continuous assemblies or conti ...

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Assembly of DNA Sequencing Data

At this writing, public databases contain the completed, contiguous sequences of a large number of bacterial genomes (e.g., http://www.tigr.org/tdb/mdb/mdbcomplete.html) (1), the yeast Saccharomyces cerevisiae genome (2); and the genomes of the nematode (3), Drosophila (4), and Arab ...

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Sequence Finishing

Sequence “finishing” is the process of turning a rough draft assembly composed of shotgun sequencing reads into a highly accurate finished DNA sequence with a defined maximum allowed error rate. The standard established by the international publicly funded sequencing community for c ...

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Quality Assessment of Finished BAC Sequences

After the sequence of a bacterial artificial clone (BAC) clone is finished, it is important to assess the completeness and accuracy of the consensus sequence that was constructed from the underlying shotgun sequencing and finishing reads. Not all sequencing projects require that the ent ...

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Exploring Transformation-Associated Recombination Cloning for Selective Isolation of Genomic Regions

Mammalian genome analysis has been advanced considerably by the development of yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) cloning systems (1,2). These techniques have made the isolation of large, random DNA fragments possible, thereby greatly s ...

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Purification of BAC DNA

The introduction of bacterial artificial chromosome (BAC) libraries has made it easy for the scientific world to gain access to an unlimited amount of DNA from different species. These BAC libraries are constructed by insertion of DNA fragments from different species into a vector, which can ...

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Hybridization-Based Selection of BAC Clones

Studying large genomic regions at the molecular level requires access to the DNA representing such sites. The availability of large and stable clones derived from target genomic regions is essential for detailed analysis such as sequencing. Since its introduction in 1992, the bacterial ...

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BAC Mapping Using Fluorescence In Situ Hybridization

The ultimate goal of the Human Genome Project is to establish the DNA sequence of human and model organism genomes as the critical first step in understanding disease, development and evolution. To accomplish this goal and a broad spectrum of applications requires integration of genome sequ ...

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Applications of Interspersed Repeat Sequence Polymerase Chain Reaction

Analysis of complex genomes includes characterization of complete large-insert genomic libraries comprising several hundreds of thousands of clones. Conventional methods to screen large-insert clone libraries for specific clones within a defined chromosomal interval ...

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High-Throughput BAC Fingerprinting

This chapter describes a nonradioactive, agarose gel-based, high-throughput DNA restriction digest fingerprinting methodology first described by Marra et al. (1) for use in the construction of high-resolution physical maps from low-copynumber, large-insert clones. The proc ...

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Amplified Fragment-Length Polymorphism and Protein Profiling for Identification of Campylobacter lari Subgroups

Amplified fragment-length polymorphism analysis (AFLP) has been shown to be a suitable method for subtyping of bacteria belonging to the genus Campylobacter. Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid ...

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Use of Peptide Nucleic Acid Probes for Rapid Detection and Enumeration of Viable Bacteria in Recreational Waters and Beach Sand

Environmental monitoring and public health risk assessments require methods that are rapid and quantitative with defined sensitivity and specificity thresholds. Although several molecular techniques have been developed to rapidly detect bacteria in complex matrices, the ...

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Detection of Legionella in Various Sample Types Using Whole-Cell Fluorescent In Situ Hybridization

The human pathogenic Legionella bacteria are found ubiquitously in natural and human-made aquatic environments as residents in biofilms, where close interactions with other microorganisms like protozoa are possible. Nosocomial legionellosis already has been linked freq ...

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Identification of Diagnostic Proteins in Mycobacterium avium subspecies paratuberculosis by a Whole Genome Analysis Approach

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne’s disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these ...

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Diagnosis of Q Fever Using Indirect Microimmunofluorescence

A microimmunofluorescence technique for the diagnosis of Q fever is described. Although this method is useful for serological diagnosis of Q fever, some technical difficulties are associated with it. First, the test antigens must be produced by a cell culture method in a level-3 biohazard fa ...

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Macrophage Cell Cultures for Rapid Isolation of Intracellular Bacteria: The Mycobacterium bovis Model

Isolation of Mycobacterium bovis from suspected cases of bovine tuberculosis demands laborious and time-consuming procedures. Also, direct PCR procedures on tissue samples show poor sensitivity, whereas radiometric and fluorescence-based identification procedures ...

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A Review of Current and Future Molecular Diagnostic Tests for Use in the Microbiology Laboratory

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined on ...

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