疾病及处理

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 ...

Construction of recombinant plasmid DNA is one of the cornerstones of molecular biology. The ability to clone DNA in a plasmid vector opens doors to downstream applications such as amplification of DNA, expression of desired genes, and construction of DNA libraries. Recombinant plasmids ...

A key step in the construction of recombinant plasmids is the verification of the successful cloning of insert DNA into the vector. A number of commonly used plasmids facilitate phenotypic selection and/or screening methods for rapid identification of insert-containing clones. Addi ...

In 1952, Joshua Lederberg coined the term plasmid to describe any bacterial genetic element that exists in an extrachromosomal state for at least part of its replication cycle (1). As this description included bacterial viruses, the definition of what constitutes a plasmid was subsequent ...

A recombinant DNA library typically represents part or all of an organism’s genomic DNA or mRNA (represented as cDNA) cloned into vectors and stored as a collection of thousands of transformants. The construction of a complete library is only half the task; researchers then need to be able to identi ...

The most widespread method used for DNA sequencing today is the Sanger dideoxy method that was first described in 1977 (1). This method takes advantage of the requirement for a free 3′ hydroxyl group to form the necessary phosphodiester bridge between two nucleotides during DNA polymerizati ...

Site-directed mutagenesis (SDM) is used to introduce a defined mutation into target DNA of known sequence to study, for example, gene expression or protein structure–function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed ( ...