遗传学实验技术

Unwinding of double-stranded DNA is required to create a single-stranded DNA template for essential DNA processes such as those involved in recombination, repair, and replication. A set of specialized enzymes called DNA helicases is dedicated to this purpose, catalyzing DNA strand sep ...

Recent advances in optical imaging and molecular manipulation techniques have made it possible to observe the activity of individual enzymes and study the dynamic properties of processes that are challenging to elucidate using ensemble-averaging techniques. The use of single-m ...

The fluorescence of the base analog 2-aminopurine (2AP) is used in highly sensitive assays to determine kinetic parameters for DNA polymerase catalyzed reactions, including exonucleolytic proofreading and nucleotide binding and incorporation. Since 2AP fluorescence can al ...

The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of rep ...

Inactivating a specific protein in vivo can yield important information about its function. One strategy previously developed inSaccharomyces cerevisiae by the Varshavsky group involves fusing a degron, derived from mouse dihydrofolate reductase, to the N-terminus of the targ ...

Temporal and spatial visualization of replication proteins and associated structures within the narrow confines of a yeast nucleus is technically challenging. Choosing the appropriate method depends upon the parameters of the experiment, the nature of the molecules to be observe ...

Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility reflects the relative ease of sample preparation and the stochiometric nature of the most popular DNA-binding dyes (propidium iodide and Sytox Green). Mammalian precedents us ...

Analysis of S phase events requires the ability to synchronize the cell cycle for subsequent physiological or molecular analysis. The cell cycle can be arrested at different stages, using drugs or temperature-sensitive mutations, and then released (block and release). These methods of ...

DNA replication takes place at discrete sites in the cell nucleus, named replication foci. The spatial arrangements of these foci change in the course of S phase in a temporally regulated and reproducible fashion forming five distinct and highly conserved replication patterns. The organ ...

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen crosslinking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach pr ...

Recent studies on genome-wide localization of transcription factor (TF) binding to DNA have shown that a large proportion of identified sequences do not contain consensus motifs predicted by databases of transcription factor binding sites, such as TRANSFAC. The main limitation of the ...

DNA binding proteins play a number of key roles in cells,in processes including transcriptional regulation,recombination,genome rearrangements,and DNA replication, repair,and modification.Of particular interest are the interactions between transcription fac ...

Effective methods of probing chromatin structure without disrupting DNA-protein interactions and associations are necessary for creating an accurate picture of chromatin and its processes in vivo. Expression of cytidine-5 DNA methyltransferases (C5 DMTases) in Saccharom ...

Nucleosome arrangements and possibly chromatin higher order structures differ in different regions of genomic DNA, and these differences could be functionally important. Nucleosome arrangements are reflected by the nucleosome ladders they give rise to upon micrococcal nucl ...

The Stanford Microarray Database (SMD) is a DNA microarray research database that provides a large amount of data for public use. This chapter describes the use of the primary tools for searching, browsing, retrieving, and analyzing data available for SMD. With this introduction, research ...

The Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) has emerged as the leading fully public repository for gene expression data. This chapter describes how to use Web-based interfaces, applications, and graphics to effectively explore, vis ...

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression by single-stranded RNAs of 18 to 25 nucleotides in length. Hundreds of miRNAs have been found in animals and plants, some of which play important roles in development or differentiation. Increasing attention has t ...

The analysis of allele-specific gene expression has been of long-standing interest in the study of genomic imprinting, but there is growing awareness that differences in allelic expression are widespread among autosomal nonimprinted genes. Recent research into cis-acting regu ...

A major challenge in systems biology is to discover and reconstruct the cis-regulatory networks through which the expression of genes is controlled. Even though a variety of sequences have been shown to interact with the transcription factors that bind DNA, extensive work is needed to disco ...

Discovery of lexical characteristics of specific sequence motifs in human genomic DNA can help with predicting and classifying regulatory cis elements according to the genes they control. In lexical models, some “words” may serve as downstream targets of signaling systems, whereas ot ...