遗传学实验技术

The dynamic modification of DNA and histones plays a key role in transcriptional regulation through �altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental s ...

Noncoding regulatory genomic elements are central for cellular function, differentiation, and disease, but remain poorly characterized. FAIRE (formaldehyde-assisted isolation of regulatory elements) has emerged as a simple method to identify and analyze active regulato ...

Binding of nuclear transactivators to sequence-specific regulatory elements on the promoter regions is of fundamental importance in gene expression and regulation. DNAbound transactivators recruit transcription coactivators or repressors and an array of associated pr ...

A subset of the proteome that binds to specific DNA sequences is at the center of genome function, integrity, and dynamics. We present a detailed protocol that allows the isolation of any specific DNA binding protein and its subsequent identification by mass spectrometry. The procedure invol ...

Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of pr ...

The aim of this chapter is to provide instruction for analyzing and mapping recent segmental and gene duplications in eukaryotic genomes. We describe a bioinformatics-based approach utilizing computational tools to manage eukaryotic genome sequences to characterize and under ...

The completion of whole genome sequencing projects offers the opportunity to exam-ine the organization of genes and the discovery of evolutionarily related genes in a given species. For the beginners in the field, through a specific example, this chapter provides a step-by-step procedure ...

Efforts to prepare a first draft of the human DNA genomic sequence forced multidisciplinary teams of researchers to face unique challenges.At the same time,these unprecedented obstacles stimulated the development of many highly innovative approaches to biomedical problem sol ...

Primed in situ labeling (PRINS) is a sensitive and specific method that can be used for the localization of single copy genes and sequences too small for detection by conventional fluorescence in situ hybridization. By the use of PRINS, the human SRY gene was localized to Yp11.31-p11.32 and the SOX3 ge ...

Comparative analysis of DNA sequences is becoming one of the major methods for discovery of functionally important genomic intervals. Presented here the VISTA family of computational tools was built to help researchers in this undertaking. These tools allow the researcher to align DNA s ...

One major goal of genomics is to identify all the functional sequences in genomes, including sequences that regulate the expression of genes. Sequence conservation is a good, albeit imperfect, guide to these functional elements. We describe how to use publicly available servers (Galaxy, t ...

Genome-wide studies are fast becoming the norm, partly fueled by the availability of genome sequences and the feasibility of high-throughput experimental platforms, e.g., microarrays. An important aspect in any genome-wide studies is determination of regulatory relationships, ...

CORG is a versatile web-based workbench for comparative promoter analysis in vertebrate model organisms. Two kinds of information are explicitly considered in the automated annotation process. First, local conservation patterns in upstream regions of homologous genes: These p ...

The existence of cell-type specific promoter and enhancer elements has been known for several years. However, the mechanisms responsible for the remarkable specificity of such elements, in comparison to the ubiquitously active promoters and enhancers of “house-keep ing” genes and DNA ...

Mutational analysis via synthetic oligonucleotides, combined with a number of biochemical approaches, has provided a wealth of information on the nature of protein-DNA interactions. Although in vitro mutational studies of the DNA target site were based initially on individual oli ...

The powerful amplification potential of PCR has assured its use in the detection of low-abundance mRNA in cells and tissues. RT-PCR is currently the most sensitive technique available for mRNA detection and quantification. It can accurately quantify genes present at only a few hundred copi ...

Substance P (SP), the most extensively studied and most potent member of the tachykinin family, is a modulator in neuroimmunoregulation (1–4). SP has been described as a peptide that is almost exclusively of neural origin (5,6). More recently, SP has been identified in non-neuronal cell types, inc ...

Although standard reverse transcriptase-polymerase chain reaction (RT-PCR) is a remarkably sensitive technique, its sensitivity can be further increased by performing “nested” RT-PCR. This involves taking an aliquot of the product from the primary RT-PCR, and using it as a template f ...

We have developed a method to detect mRNA expression in a single cell without isolating RNA from the cell (1). The method includes the following manipulations:detach cells, pick a single cell, destroy the cell by heating, reverse-transcribe RNA, and PCR-amplify. Using this method, we succeeded ...

The reverse-transcriptase-polymerase chain reaction (RT-PCR) can be used to determine minute amounts of mRNAs in tissue samples by co-amplification of a quantified amount of a competitive sequence (internal standard), so-called quantitative competitive (qc) RT-PCR. The first d ...