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丁香实验推荐阅读
Analysis of tRNA Editing in Native and Synthetic Substrates

The primary sequence of all nucleic acids in a cell contain 4 canonical nucleotides (G, A, T, and C for DNA and G, A, U, and C for RNA). However, post-transcriptionally, nucleic acids can undergo a number of chemical modifications, which may change their structure and function. tRNAs contain the most diverse ...

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Functional Analysis of Noncoding RNAs in Trypanosomes: RNA Walk, a Novel Approach to Study RNARNA Interactions Between Small RNA and Its Target

The recent discovery of thousands of small noncoding RNAs (ncRNAs), in many different organisms, has led to the need for methods to study their function. One way to help understand their function is to determine what other RNAs interact with the ncRNAs. We have developed a novel method to investigate ...

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Post-transcriptional Modification of RNAs by Artificial Box H/ACA and Box C/D RNPs

RNA-guided RNA 2′-O-methylation and pseudouridylation are naturally occurring processes, in which guide RNAs specifically direct modifications to rRNAs or spliceosomal snRNAs in the nucleus of eukaryotic cells. Modifications can profoundly alter the properties of an RNA, thus ...

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A Post-Labeling Approach for the Characterization and Quantification of RNA Modifications Based on Site-Directed Cleavage by DNAzymes

Deoxyribozymes or DNAzymes are small DNA molecules with catalytic activity originating from in vitro selection experiments. Variants of the two most popular DNAzymes with RNase activity, the 10–23 DNAzyme and the 8–17 DNAzyme, promote efficient in vitro cleavage of the phosphodiest ...

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Quantitative Detection of Epstein-Barr Virus DNA in Clinical Specimens by Rapid Real-Time PCR Targeting a Highly Conserved Region of EBNA-1

Here we describe a LightCycler-based real-time PCR for quantitative detection of EBV DNA in clinical samples such as unfractionated whole blood, serum, or plasma. This assay is based on amplification of a highly conserved 213-bp region of the EBNA-1 gene, a single-copy gene of EBV required for mai ...

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Viral Detection

The focus of this chapter is the detection of DNA viruses. The emphasis is on amplification reactions that include reverse transcription-polymerase chain reaction (RT-PCR), PCR, real-time RT-PCR, and real-time PCR methods. Amplification of the E6 and E7 oncoproteins of HPV16 is described ...

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Profiling of Epstein-Barr Virus Latent RNA Expression in Clinical Specimens by Gene-Specific Multiprimed cDNA Synthesis and PCR

We describe a two-step RT-PCR method for simultaneous detection of EBNA-1 (QK and Y3K splice variants), EBNA-2, LMP-1, LMP-2a and -2b, ZEBRA, and BARTs RNA encoded by Epstein-Barr virus. As a control for RNA integrity, the low-copy-number transcript derived from U1 A snRNP, a cellular housekeeping ge ...

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Quantitative Detection of Viral Gene Expression in Populations of Epstein-Barr Virus-Infected Cells In Vivo

The method described in this chapter uses limiting dilution analysis in conjunction with RT-PCR to determine quantitatively what percentage of EBV-infected cells within a given population are expressing the viral genes EBNA-1 Q-K, EBNA-2, LMP-1, LMP-2, BZLF-1, and the EBERs. Because this te ...

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Detection and Quantification of the Rare Latently Infected Cell Undergoing Herpes Simplex Virus Transcriptional Activation in the Nervous System In Vi

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 ...

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Reporter Cell Lines for the Detection of Herpes Simplex Viruses

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 ...

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Herpes Simplex Virus-Cell Interactions Studied by Immunogold Cryosection Electron Microscopy

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 ...

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Unraveling the Architecture of Viruses by High-Resolution Atomic Force Microscopy

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 ...

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FTIR Microscopy Detection of Cells Infected With Viruses

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 ...

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Studying the Structure of Large Viruses With Multiresolution Imaging

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 ...

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Herpes Simplex Virus-Cell Interactions Studied by Low-Fading Contrasted Immunofluorescence

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 ...

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The JC Virus-Like Particle Overlay Assay

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 ...

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Analysis of Fusion Using a Virus-Free Cell Fusion Assay

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 ...

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Use of a Real-Time, Coupled Assay to Measure the ATPase Activity of DNA Topoisomerase II

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 ...

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Analysis of Topoisomerase-DNA Interactions by Electrophoretic Mobility Shift Assay

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 ...

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Filter Binding Assays for TopoisomeraseDNA Complexes

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 ...

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