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MNase-seq, short for micrococcal nuclease digestion with deep sequencing, is a molecular biological technique that was first pioneered in 2006 to measure nucleosome occupancy in the C. elegans genome, and was subsequently applied to the human genome in 2008. Though, the term ‘MNase-seq’ had not been coined until a year later, in 2009. Briefly, this technique relies on the use of the non-specific endo-exonuclease micrococcal nuclease, an enzyme derived from the bacteria Staphylococcus aureus, to bind and cleave protein-unbound regions of DNA on chromatin. DNA bound to histones or other chromatin-bound proteins (e.g. transcription factors) may remain undigested. The uncut DNA is then purified from the proteins and sequenced through one or more of the various Next-Generation sequencing methods. MNase-seq is one of four classes of methods used for assessing the status of the epigenome through analysis of chromatin accessibility. The other three techniques are DNase-seq, FAIRE-seq, and ATAC-seq. While MNase-seq is primarily used to sequence regions of DNA bound by histones or other chromatin-bound proteins, the other three are commonly used for: mapping Deoxyribonuclease I hypersensitive sites (DHSs), sequencing the DNA unbound by chromatin proteins, or sequencing regions of loosely packaged chromatin through transposition of markers, respectively. History Micrococcal nuclease (MNase) was first discovered in S. aureus in 1956, protein crystallized in 1966, and characterized in 1967. MNase digestion of chromatin was key to early studies of chromatin structure; being used to determine that each nucleosomal unit of chromatin was composed of approximately 200bp of DNA. This, alongside Olins’ and Olins’ “beads on a string” model, confirmed Kornberg’s ideas regarding the basic chromatin structure. Upon additional studies, it was found that MNase could not degrade histone-bound DNA shorter than ~140bp and that DNase I and II could degrade the bound DNA to as low as 10bp. This ultimately elucidated that ~146bp of DNA wrap around the nucleosome core, ~50bp linker DNA connect each nucleosome, and that 10 continuous base-pairs of DNA tightly bind to the core of the nucleosome in intervals.In addition to being used to study chromatin structure, micrococcal nuclease digestion had been used in oligonucleotide sequencing experiments since its characterization in 1967. MNase digestion was additionally used in several studies to analyze chromatin-free sequences, such as yeast (Saccharomyces cerevisiae) mitochondrial DNA as well as bacteriophage DNA through its preferential digestion of adenine and thymine-rich regions. In the early 1980s, MNase digestion was used to determine the nucleosomal phasing and associated DNA for chromosomes from mature SV40, fruit flies (Drosophila melanogaster), yeast, and monkeys, among others. The first study to use this digestion to study the relevance of chromatin accessibility to gene expression in humans was in 1985. In this study, nuclease was used to find the association of certain oncogenic sequences with chromatin and nuclear proteins. Studies utilizing MNase digestion to determine nucleosome positioning without sequencing or array information continued into the early 2000s.With the advent of whole genome sequencing in the late 1990s and early 2000s, it became possible to compare purified DNA sequences to the eukaryotic genomes of S. cerevisiae, Caenorhabditis elegans, D. melanogaster, Arabidopsis thaliana, Mus musculus, and Homo sapiens. MNase digestion was first applied to genome-wide nucleosome occupancy studies in S. cerevisiae accompanied by analyses through microarrays to determine which DNA regions were enriched with MNase-resistant nucleosomes. MNase-based microarray analyses were often utilized at genome-wide scales for yeast and in limited genomic regions in humans to determine nucleosome positioning, which could be used as an inference for transcriptional inactivation. In 2006, Next-Generation sequencing was first coupled with MNase digestion to explore nucleosome positioning and DNA sequence preferences in C. elegans,. This was the first example of MNase-seq in any organism. It was not until 2008, around the time Next-Generation sequencing was becoming more widely available, when MNase digestion was combined with high-throughput sequencing, namely Solexa/Illumina sequencing, to study nucleosomal positioning at a genome-wide scale in humans. A year later, the terms “MNase-Seq” and “MNase-ChIP”, for micrococcal nuclease digestion with chromatin immunoprecipitation, were finally coined. Since its initial application in 2006, MNase-seq has been utilized to deep sequence DNA associated with nucleosome occupancy and epigenomics across eukaryotes. As of February 2020, MNase-seq is still applied to assay accessibility in chromatin. Description Chromatin is dynamic and the positioning of nucleosomes on DNA changes through the activity of various transcription factors and remodeling complexes, approximately reflecting transcriptional activity at these sites. DNA wrapped around nucleosomes are generally inaccessible to transcription factors. Hence, MNase-seq can be used to indirectly determine which regions of DNA are transcriptionally inaccessible by directly determining which regions are bound to nucleosomes.In a typical MNase-seq experiment, eukaryotic cell nuclei are first isolated from a tissue of interest. Then, MNase-seq uses the endo-exonuclease micrococcal nuclease to bind and cleave protein-unbound regions of DNA of eukaryotic chromatin, first cleaving and resecting one strand, then cleaving the antiparallel strand as well. The chromatin can be optionally crosslinked with formaldehyde. MNase requires Ca2+ as a cofactor, typically with a final concentration of 1mM. If a region of DNA is bound by the nucleosome core (i.e. histones) or other chromatin-bound proteins (e.g. transcription factors), then MNase is unable to bind and cleave the DNA. Nucleosomes or the DNA-protein complexes can be purified from the sample and the bound DNA can be subsequently purified via gel electrophoresis and extraction. The purified DNA is typically ~150bp, if purified from nucleosomes, or shorter, if from another protein (e.g. transcription factors). This makes short-read, high-throughput sequencing ideal for MNase-seq as reads for these technologies are highly accurate but can only cover a couple hundred continuous base-pairs in length. Once sequenced, the reads can be aligned to a reference genome to determine which DNA regions are bound by nucleosomes or proteins of interest, with tools such as Bowtie. The positioning of nucleosomes elucidated, through MNase-seq, can then be used to predict genomic expression and regulation at the time of digestion. Extended Techniques MNase-ChIP/CUT&RUN sequencing Recently, MNase-seq has also been implemented in determining where transcription factors bind on the DNA. Classical ChIP-seq displays issues with resoluti.... Discover the Sm Dritschilo popular books. Find the top 100 most popular Sm Dritschilo books.

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    Drama Geek

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    Most seventeen year olds don't have bucket lists, but me, Katie O'Connell doesa Wish List actually. Because I long to be someone new, the kind of girl you take notice of and rememb...