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Here we describe the use of magnetic tweezers and or microfluidics to manipulate single DNA molecules. We describe experiment which employ magnetic tweezers coupled to an inverted microscope as well as the use of a magnetic tweezers setup with an upright microscope. Using a chamber prepared via soft lithography, we also describe a microfluidic device for the manipulation of individual DNA molecules...
Cytosine methylation is the quintessential epigenetic mark. Two well-established methods, bisulfite sequencing and methyl-DNA immunoprecipitation (MeDIP) lend themselves to the genome-wide analysis of DNA methylation by high throughput sequencing. Here we provide an overview and brief review of these methods. We summarize our experience with MeDIP followed by high throughput Illumina/Solexa sequencing,...
Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K a , ΔG, ΔH, ΔS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (ΔC...
Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy...
Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the spatial patterns of interacting base pairs in known RNA structures and to reconstruct models from the collective experimental information. We focus attention on the structural context and deformability of the seven pairing patterns found...
As a result of important methodological advances and of the rapid growth of experimental data, the number of molecular dynamics (MD) simulations related to RNA systems has significantly increased. However, such MD simulations are not straightforward and great care has to be exerted during the setup stage in order to choose the appropriate MD package, force fields and ionic conditions. Furthermore,...
Our knowledge of nucleic acid structure grew rapidly over the past decade with the determination to high resolution of larger structures of great biological significance. Advances in sample preparation, crystallization techniques, cryocrystallography, access to synchrotron radiation, and crystallographic software continue to accelerate the structure determination of nucleic acids. Crystallographic...
This paper describes protocols for studies of structure and dynamics of DNA and protein–DNA complexes with atomic force microscopy (AFM) utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of...
The postgenomic era is all about learning about function by comparing genomic sequences within and between organisms. This review describes an approach that applies detailed thermodynamic information, as opposed to sequence motif searches, to analyze genomes (thermogenomics) for the occurrence of sequences with the potential to form left-handed Z-DNA and those that bind the eukaryotic nuclear factor...
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