遗传学实验技术

High-resolution fluorescence in situ hybridization (FISH) on deproteinized, stretched DNA prepared by in situ extraction of whole cells immobilized on microscope glass slides allows the visualization of individual genes or other small DNA elements on chromosomes with a resolut ...

Basic techniques for fluorescence in situ hybridization (FISH) mapping that have been used in genome projects on schistosomes and filariae are introduced. The chapter shows techniques specific for bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clo ...

Chromosome fragmentation (CF) constitutes one means of manipulating eukaryotic genomes and provides a powerful tool for examining both the structure and function of chromosomes. During the past 15 yr, CF, which is based on the use of transfection, has been widely used in yeast and mammals to elu ...

Recent advances in the field of sequencing have enabled the determination of the complete nucleotide sequence of a large number of complex genomes. The complete genome sequence of the parasite Plasmodium falciparum has been published recently, and many other parasite genome initiati ...

The chromosomes of most protozoan parasites cannot be visualized using conventional microscopy because they are too small and do not condense sufficiently at metaphase. Therefore, the development of pulsed field gel electrophoresis allowed the resolution of many parasite karyo ...

The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase ...

In recent years, a wide variety of biochemical and molecular typing systems have been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationships among different species and subspecies. Parasite sequence-spe ...

The amplified (restriction) fragment length polymorphism (AFLP) technique is a method for DNA profiling that is now widely applied for assessing diversity among various organisms with varying genomic complexity, from small bacterial to large plant genomes. AFLP analysis combines ...

Sequence information on the Trypanosoma brucei genome is rapidly accumulating. As a consequence, there is a need for techniques to analyze gene function systematically. Here, we describe a polymerase chain reaction (PCR)-based method for direct gene deletion and the generation of epit ...

The use of tyramide-coupled immunofluorescence at the single cell level provides expedient, clean, and sensitive signals for detection of DNA, RNA, or proteins. The principle is based on the ability of horseradish peroxidase (HRP) to cleave tyramides into a free radical species with a very sh ...

Immediate-early (IE) genes are the first class of viral genes expressed after primary infection or reactivation. As transcription of IE genes does not require prior viral protein synthesis, this class of genes is experimentally defined by their transcription following primary infec ...

Plasmids containing the Epstein-Barr virus (EBV) latent origin of replication, OriP, are stably maintained in human cells expressing the viral EBNA-1 protein. This stable maintenance is owing to the ability of EBNA-1 to activate DNA replication from OriP and to facilitate the segregation ...

Latent membrane protein 2A (LMP-2A) of Epstein-Barr virus (EBV) mimics a constitutively active B-cell receptor (BCR) and plays a key role in viral latency and EBV pathogenesis. By functioning as a BCR mimic, LMP-2A drives B-cell development, resulting in the bypass of normal B-cell development ...

DNA affinity purification has been used to identify cellular and viral proteins associated with the Epstein-Barr virus origin of plasmid DNA replication. This approach allows for a one- or two-step purification scheme of high-affinity DNA binding proteins from crude nuclear extracts. ...

Herpes simplex virus 1 (HSV-1) is a common and significant neurotropic human pathogen that infects 80% of all persons by adulthood. During acute HSV-1 infection, virus replicates peripherally in epithelia, enters axonal terminals, and is transported retrogradely to sensory nerve gang ...

This chapter discusses the culture of primary sympathetic neurons (superior cervical ganglia) from rat embryos and PC12 cells differentiated into neurons for use in viral infection experiments. Methods are described for the use of a neurotropic herpesvirus, pseudorabies virus (P ...

Herpesvirus genetics have long been hindered by the large size of the typical herpesvirus genome and the consequent recalcitrance of these genomes to manipulation by standard molecular genetics techniques. However, two primary strategies have emerged that allow for the generation ...

The life cycle of human papillomaviruses (HPVs) has been difficult to study in tissue culture owing to its dependence on epithelial differentiation. In this chapter several methods are described to imitate the important steps in the HPV life cycle. Normal human keratinocytes (NHKs) harve ...

Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecula genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All avai ...

The application of infectious clone technology to herpesvirus biology has revolutionized the study of these viruses. Previously the ability to manipulate these large DNA viruses was limited to methods dependent on homologous recombination in mammalian cells. However, the const ...