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丁香实验推荐阅读
Use of Dinucleotide Polymorphism Analyses in Physical Mapping

The microsatellite repeat motifs (dC-dA) n are present in high abundance in the normal genome (1). If they were to occur at regular intervals, they could be as frequent as one in approximately every 30–40 kb of the human genome. Thus, the entire genome can be represented by a large number of dC-dA or dG-dT repeat se ...

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Megaprimer PCR: Application in Mutagenesis and Gene Fusion

Since the advent of the polymerase chain reaction (PCR), a variety of PCR-based procedures of mutagenesis have been developed through the use of synthetic primers encoding the mutation. Among these, the megaprimer method and related ones (1–5) remain some of the simplest and most versatile. Va ...

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Incorporation of Thymidine Analogs for Studying Replication Kinetics in Fission Yeast

Labeling DNA during in vivo replication by the incorporation of exogenous thymidine and thymidine analogs has been a mainstay of DNA replication and repair studies for decades. Unfortunately, thymidine labeling does not work in fungi, because they lack the thymidine salvage pathway re ...

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Determining the Replication Dynamics of Specific Gene Loci by Single-Molecule Analysis of Replicated DNA

In metazoans, development and cell differentiation are known to affect various aspects of chromosomal organization at developmentally regulated gene loci (e.g., nuclear localization, locus accessibility, chromatin modifications, etc.). Recent evidence also indicates th ...

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Use of DNA Combing for Studying DNA Replication In Vivo in Yeast and Mammalian Cells

Plasticity is an inherent feature of chromosomal DNA replication in eukaryotes. Potential origins of DNA replication are made in excess, but are used (fired) in a partly stochastic, partly programmed manner throughout the S phase of the cell cycle. Since most origins have a firing efficiency b ...

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The Fast-Halo Assay for the Assessment of DNA Damage at the Single-Cell Level

The detection of breaks in mammalian cell DNA and the measurement of their repair represent primary endpoints for genotoxicity testing. Over the past three decades many techniques sensitive to the presence of DNA breaks have been developed: their availability significantly increas ...

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Monitoring Homologous Recombination Following Replication Fork Perturbation in the Fission Yeast Schizosaccharomyces pombe

Replication forks (RFs) frequently encounter barriers or lesions in template DNA that can cause them to stall and/or break. Efficient genome duplication therefore depends on multiple mechanisms that variously act to stabilize, repair, and restart perturbed RFs. Integral to at least so ...

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Computational Methods to Study Kinetics of DNA Replication

New technologies such as DNA combing have led to the availability of large quantities of data that describe the state of DNA while undergoing replication in S phase. In this chapter, we describe methods used to extract various parameters of replication — fork velocity, origin initiation rate, fo ...

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Use of DNA Combing to Study DNA Replicationin Xenopus and Human Cell-Free Systems

The Xenopus egg extract has become the gold standard for in vitro studies of metazoan DNA replication. We have used this system to study the mechanisms that ensure rapid and complete DNA replication despite random initiation during Xenopus early development. To this end we adapted the DNA combi ...

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Application of Alkaline Sucrose Gradient Centrifugation in the Analysis of DNA Replication After DNA Damage

Sucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size macromolecules is layered on the surface of a gradient whose density increases line ...

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Isolation of Restriction Fragments Containing Origins of Replication from Complex Genomes

The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of r ...

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Detection of Replication Origins Using Comparative Genomics and Recombinational ARS Assay

Effective experimental techniques are available to identify replication origin regions in eukaryotic cells. Genome-wide identification of the precise sequence elements that direct origin activity is however still not straightforward, even in the yeast Saccharomyces cer ...

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Analyzing Origin Activation Patterns by Copy Number Change Experiments

Advances in microarray technology have enabled the analysis of replication dynamics on a genome-wide scale, providing deeper insight to the factors that regulate DNA replication. Studies using high-density microarrays have led to the genome-wide identification of replication ...

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ChIP-Chip to Analyze the Binding of Replication Proteins to Chromatin Using Oligonucleotide DNA Microarrays

Chromatin immunoprecipitation (ChIP) is a widely used method to study the interactions between proteins and discrete chromosomal loci in vivo. Originally, ChIP was developed for analysis of protein associations with DNA sequences known or suspected to bind the protein of interest. The ...

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Analysis of Telomeric DNA Replication Using NeutralAlkaline Two-Dimensional Gel Electrophoresis

DNA replication studies often rely on analysis of replication intermediates, such as progressing replication forks and growing nascent strands. The assay presented here for replication at telomeres in the yeast Saccharomyces cerevisiae is based on the analysis of nascent DNA stran ...

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Topological Analysis of Plasmid DNA Replication Intermediates Using Two-Dimensional Agarose Gels

A fundamental process in DNA replication is the disentangling of the two parental strands by DNA topoisomerases. In this chapter, I detail the topological analysis of plasmid replication intermediates using two-dimensional (2D) agarose gels. The method can resolve replication int ...

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Purification of Restriction Fragments Containing Replication Intermediates from Complex Genomes for 2-D Gel Analysis

In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a ...

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Replication Initiation Point Mapping: Approach and Implications

Duplication of eukaryotic chromosomes begins from multiple sites called origins of replication, with DNA synthesis proceeding bidirectionally away from the origin. There is little detailed information available pertaining to whether replication initiates at specific si ...

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DNA Replication in Nucleus-Free Xenopus Egg Extracts

Extracts derived from Xenopus laevis eggs represent a powerful cell-free system to study eukaryotic DNA replication. A variation of the system allows for DNA replication not only in a cell-free environment, but also in the absence of a nucleus. In this nucleus-free system, DNA templates are lic ...

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High-Resolution Mapping of Points of Site-Specific Replication Stalling

Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stall ...

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