遗传学实验技术

There are many methods describing the preparation of DNA from Saccharomyces cerevisiae. The method described in this chapter is a modification of those described by Olson et al. (1) and Philippsen et al. (2). Although simpler techniques are available (3), this method routinely provides relat ...

Restriction enzyme analysis of yeast artificial chromosome (YAC)-cloned DNA allows direct comparison to genomic DNA, particularly in regions were pulsed-field gel electrophoresis (PFGE) maps are available, or to DNA cloned in other vectors (1,2). Furthermore, it allows the compari ...

The ordering of yeast artificial chromosome (YAC) clones by sequence-tagged site (STS)-content mapping has proven an effective means of constructing large, contiguously cloned arrays of DNA, some of which span almost entire human chromosomes (1). This method requires that each YAC clone ...

Fluorescence in situ hybridization (FISH) is a rapid procedure for mapping YACs on metaphase chromosomes and for identifying chimeric YACs that contain cocloned DNA fragments from different genomic regions.

The quantitative appraisal of the number of foreign gene copies integrated within the genomes of stably transfected cells is most conveniently performed using the strategy known as Southern blotting (1,2). First introduced by E. M. Southern in 1975 (2) the basic protocol involves the follow ...

The short-term expression of DNA introduced into eukaryotic cells is now widely used to investigate the biological activities of cellular and viral genes or their products. A number of different transfection methods are in common use and can be broadly divided into two categories, based on the ...

Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5, this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3) or calcium phosphate mediated gene t ...

DNA transfection is one of the most important techniques in molecular genetics. It is this technique that has made possible the dissection of complex eukaryotic genes and the characterization of the function of their components (1–7) as well as the isolation of particular genes on the basis of th ...

When DNA is introduced into eukaryotic cells, it can be integrated into the genome by homologous or illegitimate recombination 1,2. Despite great efforts to gain insight into the molecular mechanisms, our understanding of the recombination process is still in its infancy. In the absence of a m ...

Murine erythroleukemia (MEL) cell lines are erythroid progenitor cells derived from the spleens of susceptible mice infected with the Friend virus complex 1. These virally transformed cells are arrested at the proerythroblast stage of development and can be maintained in tissue cult ...

The study of gene regulation in eukaryotic cells involves a practical requirement for two distinct techniques: first, a transfection system, or more simply, a way of getting DNA into a cell; and second, a reporter system, which, as the name suggests, is a means of finding out what the transfected DNA does f ...

Efforts to expand the current repertory of cell types amenable to transfection have often been thwarted by a common obstacle-namely, the low tolerance displayed by the recipient cells toward the gene-transfer regimen itself. As a result, several laboratories have turned to the use of synth ...

Electroporation is a simple and rapid procedure by which DNA may be transferred into cells. Essentially, a high voltage pulse is applied to a suspension of cells and DNA placed between electrodes in a suitable cuvet. It is thought that this pulse induces local areas of cell-membrane breakdown, or po ...

Recent progress in techniques for the transfer of genes into cultured mammalian cells has made possible the isolation of various interesting genes from other mammalian cells, and also the study of the function and the regulation of gene expression of mammalian genes in vivo. Various transfe ...

Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Southern China. Epstein-Barr virus (EBV) infection is an important etiological factor of NPC. The fact that EBV genome is present in almost all NPC tissues renders it an ideal tumor marker for NPC. To date, quantitative analysis of p ...

Circulating RNA in plasma and serum is a newly developed area for molecular diagnosis. To date, increasing numbers of studies show that plasma and serum RNA could serve as both tumor- and fetal-specific markers for cancer detection and prenatal diagnosis, respectively. Recently, by introd ...

Mitochondrial respiratory chain disorders are clinically and genetically heterogeneous. There are several mitochondrial DNA (mtDNA) point mutations responsible for common mitochondrial diseases such as mitochondrial encephalopathy, lactic acidosis, stroke-li ...

DNA fragments from cells that have died throughout the body not only appear in the bloodstream but also cross the kidney barrier into the urine. The relatively low molecular weight (150-200 bp) of this Transrenal DNA should be considered when deciding on methods of isolation and analysis. In parti ...

This chapter describes the application of polymerase chain reaction (PCR) for the detection and quantitation of Plasmodium falciparum DNA in the plasma of malariainfected individuals. The procedure includes the following protocols: plasma sample preparation, DNA extractio ...

The etiologic agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus, known as SARS-coronavirus (SARS-CoV). Although the SARS epidemic has subsided, many authorities, including the World Health Organization (WHO) and the Centers for Disease C ...