实验基础

Specificity of primer-based amplification reactions depends on the specificity of primer hybridization. Ideally, under the elevated temperatures used in a typical amplification, the primers should hybridize only to the target sequence. However, there is a relatively narrow ran ...

Physical mapping of markers in large-insert bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones requires efficient methods for obtaining position-specific sequence information along the insert DNA. Because substantial quantities ...

The goal of the International Human Genome Project is to generate a reference sequence of the human genome with an accuracy of

Several protocols for direct sequencing of bacterial artificial chromosome (BAC) and P1 ends have been described recently (1,2). Primers located within a BAC or P1 vector have been used to sequence either end of a genomic insert to facilitate selection of minimally overlapping clones for ana ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...