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Escherichia coli Spotted Double-Strand DNA Microarrays: RNA Extraction, Labeling, Hybridization, Quality Control, and Data Management

Highly parallel hybridization of nucleic acids on glass slides has successfully been applied to measure RNA and DNA abundances in Escherichia coli (1–4). In this chapter, we summarize our experience in working with E. coli DNA microarrays accumulated over a 4-yr period. Typically, we printed a ...

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Isolation of Polysomal RNA for Microarray Analysis

DNA microarrays have been used extensively in recent years to study mRNA expression profiles of different cell types under various growth conditions. These steady-state mRNA profiles provide a wealth of information about cellular functions and responses. However, they do not necess ...

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Parallel Analysis of Gene Copy Number and Expression Using cDNA Microarrays

A cDNA microarray consists of hundreds or thousands of polymerase chain reaction (PCR)-amplified cDNAs spotted onto a glass microscope slide, in a high-density pattern of rows and columns (1). cDNA microarrays were first used widely to quantify gene expression across hundreds or thousan ...

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Genome-wide Mapping of Protein-DNA Interactions by Chromatin Immunoprecipitation and DNA Microarray Hybridization

A critical part of understanding the mechanism and logic of cellular regulatory networks is understanding where enzymes and their regulatory proteins interact with the genome in vivo. From this, we can determine the genomic features that specify protein binding and simultaneously id ...

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Statistical Issues in cDNA Microarray Data Analysis

Statistical considerations are frequently to the fore in the analysis of microarray data, as researchers sift through massive amounts of data and adjust for various sources of variability in order to identify the important genes among the many that are measured. This chapter summarizes so ...

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In Vivo DNA Analysis

The in vivo analysis of DNA-protein interactions and chromatin structure can provide several kinds of critical information regarding regulation of gene expression and gene function. For example, DNA sequences spanned by nuclease-hypersensitive sites or bound by transcription ...

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Identification of Sequence-Specific DNA-Binding Proteins by Southwestern Blotting

Southwestern blotting was first described by Bowen et al. (1) and was used to identify DNA-binding proteins that specifically interact with a chosen DNA fragment in a sequence-specific manner. In this technique, mixtures of proteins such as crude nuclear extracts or partially purified pre ...

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Site-Directed Cleavage of DNA by Linker Histone-Fe(II) EDTA Conjugates

The ordered and regular packaging of eukaryotic DNA within the chromatin complex allows the efficient utilization of this substrate for nuclear processes such as DNA replication, transcription, recombination, and repair (1,2). Thus, an understanding of the organization of protein ...

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Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots

The interaction of cell-type-specific or inducible transcription factors with regulatory DNA sequences in gene promoters or enhancers is a pivotal step in genetic reprograming during cell proliferation and differentiation and in response to extracellular stimuli. The study of ...

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Ultraviolet-Laser Footprinting

A large number of processes within the cell, in particular the regulation of gene expression, rely on the binding of proteins to specific sites on the DNA. A primary ingredient to understanding these processes is the characterization of the protein-DNA interaction (1). Such a characterizati ...

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PCR-SSCP Analysis of Polymorphism: A Simple and Sensitive Method for Detecting Differences Between Short Segments of DNA

Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) (1) is a simple method that allows one to rapidly determine whether there are sequence differences between relatively short stretches of DNA. Coupled with sequence analysis, SSCP is an extremely useful ...

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Multiplex Amplification Refractory Mutation System for the Detection of Prothrombotic Polymorphisms

First described by Newton and colleagues in 1989 (1), amplification refractory mutations system (ARMS) has become a standard technique that allows the discrimination of alleles that differ by as little as 1 bp. The system is simple, reliable, and nonisotopic. It clearly distinguishes hete ...

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Mutation Detection Using RT-PCR-RFLP

Genetic analysis by restriction fragment length polymorphism (RFLP) is one of the most common methods used to examine nucleic acids for the presence of known sequence variants. A segment that is to be searched for a mutation is amplified from genomic DNA or cDNA, digested by the appropriate restr ...

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Degenerate Oligonucleotide-Primed PCR

The amount of genomic DNA available for genetic studies can often be limiting. Degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR) is an appropriate method for overcoming these limitations by efficiently performing whole genome amplification. The DOP-P ...

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Combining Multiplex and Touchdown PCR for Microsatellite Analysis

An improved nonradioactive polymerase chain reaction (PCR)-based method for simultaneous amplification of multiple loci of microsatellites has been developed as a rapid way to screen for microsatellites (1). The approach, termed multiplex-touchdown PCR (MT-PCR), is performed ...

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Reduction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA

Repetitive sequences like short tandem repeat (STR) loci are generally referred to as slippery DNA (1). They owe this nickname to a characteristic leading to slippage within the primer-template complex during PCR elongation of the new strand (2,3), resulting in the synthesis of byproducts s ...

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Detection of Microsatellite Instability and Loss of Heterozygosity Using DNA Extracted from Formalin-Fixed Paraffin-Embedded Tumor Material by Fluores

Microsatellites are widely distributed highly repetitive DNA sequences composed of di-, tri-, or tetranucleotide repeats (1). They are spread over the whole human genome and are located between and within genes. Physiologically, they exhibit high levels of polymorphism, relative to d ...

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Three-Dimensional Reconstruction of Trypanosoma brucei Editosomes Using Single-Particle Electron Microscopy

RNA editing within the mitochondria of kinetoplastid protozoa is performed by a multicomponent �macromolecular machine known as the editosome. Editosomes are high molecular mass protein assemblies that consist of about 15–25 individual polypeptides. They bind pre-edited tra ...

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iCODA: RNAi-Based Inducible Knock-In System in Trypanosoma brucei

In vivo mutational analysis is often required to characterize enzymes that function as subunits of the U-insertion/deletion RNA editing core complex (RECC) in mitochondria of Trypanosoma brucei. The mutations may skew phenotypic manifestation of a dominant negative overexpres ...

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Perturbing A-to-I RNA Editing Using Genetics and Homologous Recombination

Evidence for the chemical conversion of adenosine-to-inosine (A-to-I) in messenger RNA (mRNA) has been detected in numerous metazoans, especially those “most successful” phyla: Arthropoda, Mollusca, and Chordata. The requisite enzymes for A-to-I editing, ADARs (adenosine deami ...

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