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丁香实验推荐阅读
Rapid Purification of DNA Topoisomerase II Containing a Hexahistidine Tag by Metal Ion Affinity Chromatography

Since DNA topoisomerases have become a major focus for scientists, the purification of these proteins from all sources represents one of the basic hurdles on the way to investigating this important class of enzymes. A variety of methods consisting of numerous steps have been applied to obtain a r ...

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Methods to Improve Efficacy in Suicide Gene Therapy Approaches: Targeting Prodrug-Activating Enzymes Carboxypeptidase G2 and Nitroreductase to Differe

Current cancer chemotherapy strategies are often hampered by the lack of tumor selectivity, resulting in unwanted damage to healthy tissue. Gene-directed enzyme-prodrug therapy (GDEPT) (1) and virus-directed enzyme-prodrug therapy (VDEPT) (2) are suicide gene therapy approac ...

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DNA-Methylation Analysis by the Bisulfite-Assisted Genomic Sequencing Method

The postreplicative methylation of DNA at the C5 position of cytosines is found in a broad spectrum of organisms ranging from prokaryotes to human (1). In prokaryotes the major role of cytosine C5 methylation (like adenine N6 and cytosine N4 methylation) is to protect the genome against DNA degra ...

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Assaying Drug-Induced Apoptosis

Apoptosis, frequently referred to as “programmed cell death” or “cell suicide,” is an active and physiological mode of cell death, where the cell itself prepares and executes the program of its own demise. A complex, multistep mechanism regulates the cell’s response to various stimuli by apopt ...

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Construction of Full-Length-Enriched cDNA Libraries: The Oligo-Capping Method

The full-length cDNA, which contains the entire sequence of the mRNA, is the ultimate goal for cDNA cloning. Unfortunately, cDNA libraries constructed by many types of conventional methods have a high content of nonfull-length cDNA clones. One of the reasons for this is that reverse transcrip ...

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Construction of Transcript Maps by Somatic Cell/Radiation Hybrid Mapping: The Human Gene Map

Systematic analysis of an organism’s transcript repertoire plays a key role in molecular genetics. The complexity of this task in higher organisms such as mammals is not only owing to the large number of expected genes (70,000–100,000) in humans (1) but also because gene coding regions are disper ...

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Preparation and Screening of High-Density cDNA Arrays with Genomic Clones

One of the greatest improvements in the use of clone libraries in genomic research was the introduction of library arrays by storing single clones in separate wells of microtiter plates. This not only makes clones practically immortal by keeping the plates at −80�C, but it also gives each of the clon ...

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Direct Selection of cDNAs by Genomic Clones

During the last decade, major advances in genomic research have resulted in the development of a variety of tools and technologies for gene identification that were successfully applied to the construction of transcriptional maps and to the identification of genes responsible for gen ...

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Exon Trapping: Application of a Large-Insert Multiple-Exon-Trapping System

Exon trapping (Fig. 1) is a technique that has been developed to identify genes in cloned eukaryotic DNA (1–7). Compared with other techniques for gene identification, exon trapping has two main characteristic features. First, it is independent of the availability of an RNA sample in which the gene ...

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Sequencing Bacterial Artificial Chromosomes

Over the last 7 years, there have been enormous changes to the methods being used to sequence DNA. These changes include the increased use of fluorescent dyes to label DNA in the sequencing reaction, improvements to automated sequencers, and the use of high-throughput methods for preparing and s ...

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Isolation of Differentially Expressed Genes Through Subtractive Suppression Hybridization

Despite the fact that the genetic blueprint in every cell is identical, an organism is composed of a multitude of different cell types. This cell type complexity or phenotypic differences are the result of differential expression of identical genes. For example, a normal cell expresses a diffe ...

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Gene Identification Using the Pufferfish, Fugu rubripes, by Sequence Scanning

The major goal of the Human Genome Project must be to identify, sequence, characterize and assign specific function to all the genes spread through our 3000 Mb of haploid DNA. Owing to the uniformity and simplicity of the DNA code, it is not an easy task to identify genes even after the region in which they lie has b ...

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Finding Genes in Genomic Nucleotide Sequences by Using Bioinformatics

You have, we hope, come to this chapter wishing to know how to use bioinformatics to locate genes in genomic sequences. You probably want to know which are the easiest and best tools to use, to treat them largely as black boxes, and to know how to assess the likely accuracy of the results you obtain. If so, you have come to t ...

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Construction of Microsatellite-Based, High-Resolution Genetic Maps in the Mouse

The mapping of genes underlying either simple mendelian or complex traits can be broken down into a number of distinct stages—initial detection of the locus in a genome scan, determination of the most likely map location for the gene, and finally fine mapping of the locus. A number of experimental str ...

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Isolation of Differentially Expressed Genes by Representational Difference Analysis

Representational difference analysis of cDNA (cDNA RDA) is designed to compare two different mRNA populations resulting in the isolation of differentially expressed genes. RDA was originally developed for genomic DNA as a method to isolate the differences between two complex genom ...

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Expression Profiling by Systematic High-Throughput In Situ Hybridization to Whole-Mount Embryos

The genome of a given organism is considered in biology as the fundamental invariant (1). It is virtually the same throughout lifetime and, to a lesser extent, over generations. By contrast, genetic information is expressed in complex and ever-changing temporal and spatial patterns throug ...

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Expression Profiling and Isolation of Differentially Expressed Genes by Indexing-Based Differential Display

Profiling the expression status at the level of transcription is a starting point for delineation of the function of both known and unknown gene products. In addition, the identification of genes expressed in a regionally, temporally, or environmentally specific manner is fundamental to ...

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Expression Monitoring Using cDNA Microarrays: A General Protocol

As the Human Genome Project nears the completion of the first human sequence, the next great challenge is to elucidate the function of these genes. One route of exploring the function of a gene is by determining its pattern of expression. Various methods are available for detecting and quantitati ...

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Identification of Novel Genes by Gene Trap Mutagenesis

Functional analysis of the mammalian genome will be a major task of biologic science in the future. Gene trap is a method designed to provide functional information for novel genes (1–3). It offers tagging of a gene (facilitating subsequent cloning), analysis of the expression pattern by a simple s ...

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Prediction of Protein Structure and Function by Using Bioinformatics

Proteins mediate virtually all biological processes. Understanding the mechanisms by which proteins function requires a knowledge of their three-dimensional (3D) structures. As a consequence of the genome and full-length cDNA sequencing projects, there are several orders of ma ...

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