疾病及处理

The isolation of high-quality genomic DNA is often the initial step for molecular genetic analyses and crucial to many applications within the molecular research of malaria. Criteria for quality of DNA are purity, stability, and integrity and size of the molecules. Quantity also often is an im ...

The isolation of RNA and its subsequent characterization by various applications, such as the generation of cDNA libraries, Northern blot analysis, and reverse transcriptase polymerase chain reaction studies (RT-PCR), are fundamental for understanding the mechanisms underl ...

Southern blotting, first described by E. M. Southern in 1975 (1), is one of the cornerstones in molecular biology. The idea to immobilize DNA on a solid support was first proposed by Denhardt (2). Based on this, methods were developed for the identification of specific sequences in dot blots and recombi ...

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are complementary methods for separating and detecting the presence of a specific protein from a complex mixture. Proteins, from a cell extract, for example, are separated electrophor ...

In the natural and untreated intermediate hosts, Plasmodium infections are maintained for extended and often lifelong periods. Parasitemia generally reaches its highest level in the first peak after inoculation, and only rises thereafter during increasingly brief and intersp ...

To achieve a more complete understanding of malaria parasite biology and its interaction with its host, it is essential to be able to study the transcription and expression of parasite genes. Northern blot analysis and RNase protection assays are commonly used to study gene expression (1). How ...

The genetic identification of malaria parasites has become an important task for epidemiological studies and for laboratory-based studies (1–7). A large number of adapted field isolates have been cultured and widely circulated during the past years. However, only few of these have been cl ...

The technique of in situ RNA hybridization provides a means of examining RNA expression within individual parasites at many stages of development in both the vertebrate and mosquito hosts. The protocols described in this chapter have been developed with the aim of preserving the morpholo ...

Sequencing of the entire genome from the human malaria parasite, Plasmodium falciparum, began in earnest in 1996 with the formation of an international consortium of scientists and funding agencies (1). Due to the instability of the highly adenine (A) and thymine (T)-rich P. falciparum DNA in la ...

Genomic DNA libraries represent the total complement of the genetic information of an organism’s DNA, as opposed to cDNA libraries, which contain only the protein encoding sequences expressed at a particular stage of the life cycle. Ideally, a genomic library contains the coding and control ...

Mung bean nuclease (MBN) has been used typically for its single-stranded nuclease activity (1). Under defined reaction conditions, however, which include altered solvation and elevated temperature, hypersensitive sites surrounding coding regions become sensitive to nucle ...

cDNA libraries represent the genetic information encoded in the messenger RNA (mRNA) of a particular tissue or organism. Construction of a cDNA library begins with the isolation of purified and full-length RNA. This isolation is made difficult by the fact that RNA molecules are exceptional ...

Subtractive hybridization techniques are designed to deplete one cDNA population (the “target”) of sequences common to a second group of cDNAs (the “driver”), thereby effectively enriching the target population for unique sequences. The applications of this powerful methodology ...

The discovery by Burke et al. in 1987 (1) that yeast can accept large pieces of heterologous DNA as yeast artificial chromosomes (YAC) marked a milestone in the analysis of complex genomes. While standard prokaryotic cloning systems, such as plasmids and cosmids, are limited by the size of the DNA frag ...

Genetic transformation of an organism is a fundamental investigational tool that allows the researcher to gain an insight into the basic cellular biology. Numerous areas of parasite biology can be addressed simply by introducing plasmids that are maintained as episomes into the cell of c ...

Gene targeting, the technology that permits inactivation or modification of a gene by homologous recombination, has been reproducibly established in two plasmodial species: Plasmodium falciparum (1), the most important pathogen for humans, and Plasmodium berghei (2), which infe ...

Vaccination is the most cost-effective measure to control the pathological effects of an infectious agent (1,2). With the combined use of molecular cloning and of modern sequencing methods, the sequence of many protein components present in different infectious agents have been eluci ...

There is increasing recognition that the unique antigenicities of the different stages of the Plasmodium spp. life cycle, the requirement for distinct immune mechanisms targeting these different stages, the immense allelic variation of parasites in the field, and the genetic hetero ...

To determine the immunogenicity of vaccine candidates, it is essential to establish an accurate and convenient methodology for assessing the level of antigen-specific T-cell responses, and several methodologies have been developed. These include the T-cell proliferative assay ...

In all rodent models of erythrocytic-stage malaria infections, the T-cell response to the parasite has been shown to be crucial for the development of protective immunity (1-5) and in some cases is thought to play a role in the pathology of malaria (6,7). To determine which effector functions of T cells m ...