遗传学实验技术

Herpes simplex virus (HSV), in contrast to most other members of the herpes virus family, has the ability to infect, enter latency, and reactivate from latency in a number of nonhuman species, including mice. This provides a unique opportunity to study the complex lytic-latent cycle of a human neuro ...

Virus culture has played significant roles in basic and clinical virology, with a number of advantages that cannot be attainable by modern molecular techniques. However, virus culture is generally a slower process, as it inevitably takes the period of a full replication cycle of a given virus. A g ...

A technique is presented for high-resolution postembedding immunolocalization of one or two (or several) antigens in the same ultrathin cryosection using primary monoclonal antibodies from the same species. The optimized three-layer indirect immunogold-labeled cryosec ...

Atomic force microscopy (AFM) has recently emerged as an effective complement to other structure determination techniques for studying virus structure and function. AFM allows the direct visualization of viruses in a hydrated state and can probe surface topography in unrivaled det ...

Fourier-transform infrared (FTIR) microscopy is considered a comprehensive and sensitive method for detection of molecular changes in cells. The advantage of FTIR microspectroscopy over conventional FTIR spectroscopy is that it facilitates inspection of restricted regi ...

Multiresolution imaging is an extremely useful technique for understanding in detail the struc ture of large DNA viruses that do not yield to the requirements of protein crystallography. The methodology consists in fitting the atomic structures of capsid components, independently ...

The low-fading immunofluorescence with propidium iodide contrast described here is recommended for light and confocal viral antigen identification and other cell biology studies because: (1) it is a simple, rapid, sensitive, and reproducible technique; (2) phase-contrast micr ...

JC virus (JCV) belongs to the family of double-stranded DNA polyomaviruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML). It has been reported that sialic acids play a pivotal role in hemagglutination of r ...

For enveloped viruses, such as viruses within the herpesvirus family, of which Epstein-Barr virus (EBV) is a member, infection of target cells includes two distinct steps. The first is characterized by the binding of viral envelope glycoproteins to host cellular receptors. After binding, t ...

This chapter describes the use of a common spectrophotometric assay for following the rate of ATP hydrolysis as applied to type II DNA topoisomerases. It is called a “coupled assay,” because each time an ATP molecule is hydro-lyzed, a molecule of NADH is rapidly oxidized; ATP hydrolysis and NADH oxid ...

DNA topoisomerases break and rejoin DNA strands through a covalent pro-tein-DNA intermediate. The reaction chemistry involves nucleophilic attack by a tyrosine moiety of the enzyme on the phosphodiester backbone of DNA to form a phosphotyrosyl linkage to one (in the case of type I enzymes) or ...

Protein-nucleic acid interactions have been studied by density shift gradients, affinity columns, and gel retardation. Some of these methods are tedious or have severe limitations in quantitative analysis. Filter binding assays are fast, sensitive, and suitable for physical chem ...

Type I topoisomerases catalyze the reversible nicking of duplex DNA (for review, see ref. 1). In the nicked state, the enzyme is attached to the DNA by way of a phosphodiester bond between a tyrosine residue in the protein and the end of the broken strand. The polarity of attachment depends on the source of the en ...

Serial analysis of gene expression (SAGE™) is a patented, large-scale mRNA-profiling technology that produces comprehensive, quantitative, and reproducible gene expression profiles (originally described in refs. 1 and 2). Unlike the alternative technologies of differenti ...

Much of modern genetics is based on analysis of DNA sequence. Therefore, there is great pressure to scale up sequence analysis while decreasing its cost. The most promising platforms are based on the use of oligonucleotide arrays (DNA chips), which perform many analyses in parallel (1). Arrays co ...

RNA arbitrarily primed polymerase chain reaction (RAP-PCR) has been used extensively to identify differentially regulated genes (1–6). The RAP-PCR method begins with conversion of RNA into cDNA, followed by arbitrarily primed PCR. The technique uses arbitrarily primed PCR (7–11) to am ...

Differential display of mRNA via polymerase chain reaction (DD-PCR) has become a powerful procedure for the quantitative detection of differentially expressed genes in distinct cell populations (1–4).

AU-rich elements (AREs) are found in 3′ untranslated regions (3′ UTR) of many highly unstable mRNAs for mammalian early-response genes. The minimal AU sequence core within the ARE is the heptamer WAUUUAW, although from a functional point of view, several pentanucleotides clustered in close p ...

Since the completion of the human genome-sequencing project, scientists are now able to read the code of all human genes stored on the 46 chromosomes of the human genetic library. However, we are far from reaching an understanding of the functional relationships existing between more than a tiny f ...

Real-time quantitative polymerase chain reaction (PCR) methods (1) can be further optimized for various purposes by performing quantitative PCR for multiple RNA species in one sample (2). Advantages of this method not only include the elimination of differences in reaction mix volumes ...