Springer protocols feed by Genetics/Genomics 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Single-Molecule Approaches for the Characterization of Riboswitch Folding Mechanisms
Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformatio...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Southwestern Blotting Assay
Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography. A blot resulting from 1-dimensional SDS-PAGE reveals the molecular weight of the binding proteins. To increase separation and determine isoelectric point a 2-dimensional gel can be blotted. Additional dimensions of electrophoresis, such as a gel shift (EMSA), can precede isoelectric focusing and SDS-PAGE to further improve separation. Combined with other techniques, such as mass spectrometry, the DNA-b...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
In Cellulo DNA Analysis: LMPCR Footprinting
The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR,...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Determining the Architecture of a Protein–DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program
Determining the structure of a protein–DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relati...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
In Vitro DNase I Footprinting
The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky nucleases such as DNase I. The DNase I footprinting method was developed to take advantage of this fact in the study of DNA-protein interactions: it consists in comparing the pattern of fragments generated by the partial digestion of a DNA sequence in the absence of a protein to that produced by its partial digestion in the presence of said protein. Normally, when the two sets of fragments are separated side by side on a gel, the ladder of DNase I-generated fragme...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Identification of Nucleic Acid High Affinity Binding Sequences of Proteins by SELEX
A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein–nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Quantitative Investigation of Protein–Nucleic Acid Interactions by Biosensor Surface Plasmon Resonance
Biosensor-surface plasmon resonance (SPR) technology has emerged as a powerful label-free approach for the study of nucleic acid interactions in real time. The method provides simultaneous equilibrium and kinetic characterization for biomolecular interactions with low sample requirements and without the need for external probes. A detailed and practical guide for protein–DNA interaction analyses using biosensor-SPR methods is presented. Details of SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples, experimental design, quantitati...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes
Electrophoretic mobility shift assays (EMSA) have proven their usefulness for studying interactions between biological molecules. In the present protocol, a purified protein of interest is mixed with a 5′-end radiolabeled DNA probe. The bound complexes are separated by electrophoretic migration through a polyacrylamide gel and detected with a phosphorimager. The applications of EMSA are diverse, from thermodynamic and kinetic analyses to observation of bending and other conformational changes, stoichiometric inferences, or insights into cooperative protein binding. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Circular Dichroism for the Analysis of Protein–DNA Interactions
The aim of this chapter is to provide information on the practical aspects of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) in protein–nucleic acids interaction solution studies. The chapter will describe the guidelines appropriate to designing experiments and conducting correct data interpretation, the use of both benchtop and synchrotron CD approaches is discussed and the advantages of SRCD outlined. Further information and a good general review of the field a can be found in Gray (Circular Dichroism of protein–nucleic acid interactions. In: Fasman GD (ed) Circular dichroism and ...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Aggregate and Heatmap Representations of Genome-Wide Localization Data Using VAP, a Versatile Aggregate Profiler
In the analysis of experimental data corresponding to the signal enrichment of chromatin features such as histone modifications throughout the genome, it is often useful to represent the signal over known regions of interest, such as genes, using aggregate or individual profiles. In the present chapter, we describe and explain the best practices on how to generate such profiles as well as other usages of the versatile aggregate profiler (VAP) tool (Coulombe et al., Nucleic Acids Res 42:W485–W493, 2014), with a particular focus on the new functionalities introduced in version 1.1.0 of VAP. (Source: Springer protocols ...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Global Mapping of Open Chromatin Regulatory Elements by Formaldehyde-Assisted Isolation of Regulatory Elements Followed by Sequencing (FAIRE-seq)
Genetic information is organized in a complex structure composed of DNA and proteins together designated chromatin. Chromatin plays a dynamic role in transcriptional processes in that alteration of the interaction between its components results in the deregulation of cellular transcriptional program. Modification of epigenetic marks, variation in the precise positioning of nucleosomes, and consequent mobilization of nucleosomes regulate the access of various transcriptional factors to its underlying DNA template. Nucleosome-depleted regions, also designated open chromatin domains, are associated with active DNA regulatory ...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Detection of Short-Range DNA Interactions in Mammalian Cells Using High-Resolution Circular Chromosome Conformation Capture Coupled to Deep Sequencing
DNA interactions shape the genome to physically and functionally connect regulatory elements to their target genes. Studying these interactions is crucial to understanding the molecular mechanisms that regulate gene expression. In this chapter, we present a protocol for high-resolution circular chromosome conformation capture coupled to deep sequencing. This methodology allows to investigate short-range DNA interactions (<100 kbp) and to obtain high-resolution DNA interaction maps of loci. It is a powerful tool to explore how regulatory elements and genes are connected together. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Selection and Validation of Spacer Sequences for CRISPR-Cas9 Genome Editing and Transcription Regulation in Bacteria
We describe a procedure involving computational and experimental steps to identify and test potentially interesting spacer sequences in bacterial genomes. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Chromatin Endogenous Cleavage (ChEC) as a Method to Quantify Protein Interaction with Genomic DNA in Saccharomyces cerevisiae
Chromatin endogenous cleavage (ChEC) is a technique which allows to monitor protein-DNA interaction in the nucleus of eukaryotic cells. In addition to mapping of genomic interaction sites ChEC may also yield quantitative information about the occupancy of proteins at their genomic target regions. Here, we provide a protocol for ChEC experiments in S. cerevisiae, downstream DNA analysis and quantification of ChEC-mediated degradation. The potential of the method is exemplified in ChEC experiments with RNA polymerase I and the yeast homolog of linker histone H1. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
Individual and Sequential Chromatin Immunoprecipitation Protocols
DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene expression making it relevant to determine the profiles of cohabitation of several proteins on the chromatin fiber. Chromatin immunoprecipitation (ChIP) has been broadly used to determine the profile of several histone posttranslational modifications as well as transcription factor occupancy in vivo. However, individual ChIP does not resolve whether the epitope under study is present at the same time on a given genomic location. Here we describe the ChIP...
Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news
