实验基础

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separates proteins by their iso-electric points (IEPs) and electro-phoretic mobilities (1). In the first dimension, a sample is applied to an iso-electric focusing (IEF) acrylamide gel. Within this the con stituent pr ...

DNA transfection is the introduction of molecular constructs into parasites in a manner permitting the expression of encoded genes. Transfection is an exceedingly powerful technique for analyzing gen etic regulatory elements and gene function, and is widely used in molecular biolo ...

Physical mapping of protozoan chromosomes has been transformed in recent years by the introduction of pulsed field gradient electrophor- esis (PFGE) (1). The chromosomes, which otherwise are too small to be detected in condensed form or mapped by conventional in situ hybridization meth ...

One of the most powerful ways of identifying genes is to utilize oligonucleotide probes. A prerequisite for this approach is the exist ence of a minimal amount of protein sequence information, either from the parasite protein itself (see Chapter 33) or from homologous proteins of other organi ...

This chapter essentially combines a variety of techniques already described in other chapters to allow a rapid characterization or con firmation of λ expression clones. Antibodies used to screen expres sion libraries (see Chapter 21) may have been raised against affinity purified anti ...

The many and various species of Leishmania are responsible for a broad spectrum of human and animal diseases known collectively as the leishmaniases. They are widely distributed in the warmer parts of the world and transmitted by the bite of infected female phlebotomine sandflies. The life cy ...

Over the last decade major advances in cloning technology have markedly improved the ability to construct clone libraries expressing foreign proteins and to screen them to identify clones encoding any desired gene product. A number of types of libraries, expression vec- tors, and screeni ...

The phylogeny of a large number of parasite species is still entirely based on morphological and physiological features. However, the reliability of such criteria becomes highly questionable, especially for species exhibiting a low level of organismal complexity. The advent of mode ...

Nucleic acid hybridization probes have a wide range of applica tions for the detection, identification, and quantification of microor ganisms, from environmental studies to medical diagnoses (1,2). They offer unique advantages in terms of sensitivity and specificity, with their po ...

The relationship between host and parasite is a dynamic one with both groups undergoing genetic change to maximize reproductive success. The short generation time and high reproductive capacity of the malaria parasite provides scope for extensive mutation within the parasite geno ...

The polymerase chain reaction (PCR) allows the specific amplifica tion of either RNA or DNA nucleotide sequences (1,2). The hallmarks of this technique are specificity, sensitivity, and speed. The specific ity of the reaction is a result of the requirement of DNA polymerases for a primer that is ex ...

Detection of malaria parasites to as low a level as one organism is essential for epidemiological surveillance and effectiveness of thera peutic treatments. Classical detection of the parasites has always relied on microscopic examination, a method that is relatively simple and inex ...

Parasitic protozoa, generally, present as highly disperse popula tions with complex life cycles. The ability to attain and sustain this diversity and complexity resides in the DNA of the cell. Although there is a great deal of activity directed toward analysis of parasitic protozoan DNA at the m ...

The use of DNA probes for the detection and identification of para sites has become a routine practice in many laboratories and the tech nique is rapidly becoming established as a diagnostic tool for field and clinical studies. DNA probes have been used in the identification of a large number of paras ...

Whole parasites or nucleic acids from these organisms can be immo bilized on filter supports, such as nylon or nitrocellulose. This allows many samples to be tested for the presence of a specific sequence by hybridization to a complementary DNA probe. In addition, the copy number of this sequence can ...

Trypanosoma cruzi is a protozoan flagellate that is transmitted to mammals by bloodsucking triatomine bugs. Transmission is not by the bite of the insect but by contamination of skin abrasions or mucous membranes with bug feces containing infective (metacyclic) trypomastigote form ...

The preparation of nucleic acids from schistosomes can be divided into several stages, beginning with the collection and cleaning of the parasite material, its subsequent mechanical disruption to release cellular contents, the separation of nucleic acids from that lysate and, final ...

Extraction of nucleic acids is fundamental to molecular genetic studies of parasitic organisms. DNA is required for gene bank construction and analysis of the genome, and RNA is needed for cDNA synthesis, analysis of transcription, and translation studies. Early methods of isolating DNA ...

The genus Leishmania includes species that are the causative agents of visceral, cutaneous, and mucocutaneous leishmaniasis. Infections caused by these organisms are common in both the Old and New Worlds, where they represent a major public health problem. As a result, Leishmania species ...

The World Health Organization estimates that between 16 and 18 million people are infected with Trypanosoma cruzi, the protozoan parasite that causes American trypanosomiasis, or “Chagas′ disease” (1). An additional 70 million people are thought to be at risk of infection with the parasit ...