实验动物总则

Hepatitis C virus (HCV) was identified as a major causative agent of non-A, non-B hepatitis (1). Numerous complete or partial nucleotide sequences of HCV isolates have been reported worldwide, and comparison of these sequences revealed their marked genetic heterogeneity nature, sugge ...

Hepatitis C virus (HCV), a flavi-like virus with a positive-sense, single-stranded genome RNA (1–3), shows considerable variation in nucleotide sequences. These variations fall into a series of specific patterns and are the basis for classification of HCV into different types and genoty ...

Several different methods have been developed for the typing of HCV variants: direct sequence analysis, slot-blot hybridization analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products using cDNA probes specific to each HCV genotype and PCR amplificati ...

Hepatitis C viruses (HCVs) constitute a highly variable genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes a polyprotein which is co- and posttranslationally cleaved into at least nine prot ...

The RNA genome of hepatitis C virus (HCV) displays extensive sequence variation, and consequently, the virus is classified into six major genotypes. The severity of disease and response to antiviral treatment are thought to be influenced by both viral and host-related factors, including a ...

Most RNA viruses exists as a heterogeneous mixture of closely related viral genome in the host, which is the result of high error rates in RNA replication and selected by various viral and host factors. The spectrum of this spectrum of related genomes within a host is referred as quasispecies (1). This im ...

The heteroduplex tracking assay (HTA) is a tool that can be used for determining genotype, quasispecies analysis, molecular evolution, and epidemiological studies (1–7). By hybridizing a labeled, single-stranded DNA probe to colinear, reverse transcriptase (RT) PCR products from a s ...

Hepatitis C virus (HCV) is believed to replicate via a viral-encoded, RNA-dependent RNA polymerase. This replication strategy has limited fidelity Thus, HCV is genetically heterogenous. To date, six HCV genotypes and more than 80 viral subtypes have been identified. Further, even within i ...

Hepatitis C virus (HCV) is the agent responsible for the majority of cases of the parenterally transmitted non-A, non-B hepatitis. The major obstacles to its discovery were the low level of replication in the infected host, both natural and experimental, and the low immunogenicity of its protei ...

A reliable detection system for HCV antigens in liver tissue may be used to identify the HCV cellular tropism and subcellular sites of viral replication Also, it can be used to study the relationship between viral expression and disease activity. Finally, it can facilitate the study of host-viral ...

The procedure described below was originally reported to detect the hepatitis C virus RNA (genomic strand) by nonradioisotopic in situ hybridization in formalin-fixed, paraffin-embedded liver tissue of two acutely infected chimpanzees, in a collaborative study conducted with R. ...

The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations require the isolation of high-purity plasmid DNA. Commercial anion-ex ...

Plasmid extraction is typically performed to produce template DNA for a desired molecular biological reaction, or set of reactions, such as restriction endonuclease digestion (see Chapter 20), DNA sequencing (see Chapter 22), in vitro mutagenesis (see Chapters 23–26), transformat ...

Cosmid and bacterial artificial chromosome (BAC) systems have been developed for the cloning of large DNA inserts averaging 40 kb and 130 kb (range: 90–300 kb), respectively. The resulting clones are more stable than yeast artificial chromosomes (YACs) and rarely chimeric, which makes them e ...

Single-stranded DNA (ssDNA) is the optimal template for most polymerase-based molecular-biology applications, including DNA sequencing and site-directed mutagenesis. Phagemids are chimeric vectors, derived from the ssDNA bacteriophages M13, fd, or f1, that normally repli ...

Manipulation and analysis of DNA sequences is often a complex task involving many steps, each of which must be carefully planned and executed. To facilitate this process, the number of steps should be minimized and each step analyzed to ensure that it has been completed successfully. Often, this a ...

A fundamental step in molecular biology is the cloning of a DNA fragment insert into a plasmid vector. This allows the cloned fragment to be replicated upon transformation of the recombinant molecule into a bacterial cell (see Chapters 4 and 5) so that the DNA of interest can be investigated further. C ...

A common step in cloning experiments is the purification of DNA fragments prior to ligation. Often, both the insert and vector DNA fragments will be derived from restriction endonuclease digests and, thus, will be mixed with enzymes, salts, and possibly other DNA fragments that may inhibit the en ...

Since it was described in 1988 (1), the polymerase chain reaction (PCR) has been a valuable tool for molecular biologists. PCR allows researchers to produce a large quantity of a desired DNA fragment while requiring only a small amount of template. Prior to PCR, isolation of DNA fragments was typical ...

Lambda (λ) bacteriophages are viruses that specifically infect bacteria. The genome of λ-phage is a double-stranded DNA molecule approx 50 kb in length (1). In bacterial cells, λ-phage employs one of two pathways of replication: lytic or lysogenic. Commonly, λ-phage vectors replicate via the l ...