遗传学实验技术

In health, approx 4% of cytosines are methylated. Tissues can vary in their levels of DNA methylation and the overall level is often reduced in malignancy (1). The level of DNA methylation is usually obtained by chromatographic separation of the constituent nucleotide bases or their related de ...

The presence of 5-methylcytosine as a modified base in DNA was discovered many decades ago. Surprisingly, however, and despite intense research efforts, the principal function of DNA methylation is still unknown. The CpG dinucleotide is the predominant if not exclusive target sequence f ...

Methylation of cytosine residues is an almost ubiquitous finding of higher organisms (1). The majority of this methylation occurs at the dinucleotide CpG (where p denotes a phosphate group) (2). CpG sites are distributed throughout the genome with clusters of the sequence being found in the 5′ pr ...

Restriction landmark genomic scanning (RLGS) is a method that provides both a quantitative genetic and epigenetic (cytosine methylation) assessment of thousands of CpG islands in a single gel without prior knowledge of gene sequence (1). The method is a two-dimensional separation of ra ...

Most molecular biological techniques used to analyze specific loci in complex genomic DNA involve some form of sequence-specific amplification, whether it is biological amplification by cloning in Escherichia coli, direct amplification by polymerase chain reaction (PCR), or s ...

Differential hybridization and its related techniques have been developed to identify genes whose expressions are altered during different physiological conditions or in dissimilar cell or tissue types (1–3). High-throughput DNA array technologies have further advanced th ...

CpG islands are clusters of CpG dinucleotides that can be found in the 5′ region of about half of human genes (1). Methylation of cytosine within the 5′ CpG islands is associated with transcriptional inactivation of the involved gene. Aberrant methylation of CpG islands is an important mechanism of ...

Inoculation of plasmid DNA vectors encoding immunogenic proteins induce humoral as well as cell-mediated immune responses. Protection against challenge with pathogens has provided protective immunity in several instances in animal models. (1,2). DNA vaccines allow the simult ...

Within the last five years, the exponential growth of research activities on the development of genetic vaccination and gene therapy has made it necessary to develop an easy, cost-effective, industrial scale process for production of plasmid DNA (see Note 1). One main issue is that the process sh ...

The use of DNA vectors to elicit an immune response has produced a lot of interest. Unfortunately, one of the limiting factors has been the problem of gene expression. In order to obtain a strong expression of the vaccinating gene, several steps are necessary. The vector has to be delivered in such a way that it ...

DNA-based vaccination is a potent technique to prime cellular (T-cell mediated) immune responses (reviewed in 1). Many details of the priming of T-cell precursors by antigen translated from injected expression plasmid DNA are unknown. The relevant cell that is transfected in situ after DNA ...

Intramuscular injection of naked plasmid DNA is known (1–3) to elicit humoral and cell-mediated immune responses against the encoded antigen. It is thought (2,3) that immunity follows DNA uptake by muscle cells, leading to the expression and extracellular release of the antigen which is then ...

Particle-mediated delivery involves coating materials onto the surface of dense sub-cellular sized (0.5–5 mm) particles and accelerating the particles to sufficient velocity to penetrate target cells. The technique was invented by Sanford and Wolf at Cornell University (1) to tran ...

The mucosal surface area of the gastrointestinal, genitourinary and respiratory tracts is more than 200 times greater than that of the skin and is the primary site of transmission of numerous diseases. The entry of pathogenic organisms at mucosal surfaces can be prevented by mucosal, but not sy ...

Intercellular signaling and cell adhesion are among the most critical mechanisms for development and maintenance of multicellular organisms. Although a large number of molecules involved in signaling or adhesion have been cloned, there still remain many unknown molecules that are ...

The budding yeast Saccharomyces cerevisiae has been a valuable model in establishing eukaryotic DNA topoisomerase I as the cellular target of specific antineoplastic agents, including camptothecin (1–3), aclacinomycin A (4), and R-3, a rebeccamycin analogue (Vance, J. R., Woo, M. H., Otero, A ...

Eukaryotic DNA topoisomerase I is a highly conserved enzyme that catalyzes the relaxation of positively and negatively supercoiled DNA (1–4). The enzyme binds duplex DNA and transiently cleaves a single DNA strand. This is accompanied by the formation of a phospho-tyrosyl linkage betwe ...

Integral to all catalytic functions of topoisomerase II is the ability of the enzyme to generate transient double-stranded breaks in the backbone of DNA (1–3). When topoisomerase II cleaves DNA, it maintains the topological integrity of the genetic material by forming covalent bonds betw ...

Type II topoisomerases act by making a double-stranded break in a DNA segment, passing an intact DNA segment through the break, and resealing the break 1–4). The DNA strand passage reaction of topoisomerase II can be either intra- or intermolecular. The enzyme relaxes supercoiled DNA in an intramo ...

Numerous type I and type II DNA topoisomerase genes have now been identified and sequenced from both the eukaryotic and prokaryotic sources. Amino acid sequence alignments of these genes firmly establish that all type II topoisomerase genes belong to one family regardless of source, where ...