微生物学技术

Celiac disease is an immune-mediated disorder that primarily affects the small intestinal mucosa. It is one of the few human disorders of which it is possible, and ethically acceptable, to obtain samples from the disease-affected tissue. This chapter describes how small intestinal biopsy ...

Large-insert genomic DNA libraries are based on the Escherichia coli F factor, a low-copy plasmid that exits in a supercoiled circular form in the host cells. These libraries are used to provide a way to divide complex genomes into DNA segments, thereby reducing the complexity (1). The libraries are ...

Recent advances in the Human Genome Project have opened the door to new approaches in biologic research. The availability of the human draft sequence (1) now offers the tool for sequence-based genetic analyses on a genomewide level. However, owing to the fragmented and incomplete nature of the d ...

Shotgun cloning is a method to generate the templates needed for DNA sequencing. This process entails breaking a large target DNA randomly into smaller fragments; end sequencing these smaller fragments; and from the overlapping sequences of the randomly generated fragments, reassem ...

Robust and reproducible isolation of high-quality templates is a requirement for successful DNA sequencing. To date, approaches for template generation have been limited to purification of biologically propagated M13 or plasmid-based templates, or in vitro amplification of su ...

Transposon-mediated sequencing is an effective method for obtaining full-length high-quality DNA sequence. This method can be applied to the finishing stages of bacterial artificial chromosome (BAC) sequencing, allowing the user to expand a finishing repertoire of custom oligo ...

Pyrosequencing, a bioluminometric DNA sequencing technique based on sequencing by synthesis, is emerging as a widely applicable tool for detailed characterization of nucleic acids (1–3). This technique relies on the real-time detection of inorganic pyrophosphate (PPi) releas ...

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 ...