chemistry

MNase-seq

MNase-seq is a chemistry topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand MNase-seq rather than just read about it. In short: 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.

Key takeaways

MNase-seq belongs to chemistry; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect MNase-seq to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of MNase-seq from memory before moving on to harder problems.

Reference excerpt

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.

… excerpt ends here. Continue reading the full article.

Illustrations

Worked examples

Example 1 — a first encounter with MNase-seq

Start with the simplest possible case. Write down what MNase-seq claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In chemistry, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to MNase-seq before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about MNase-seq ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of MNase-seq

In research: MNase-seq appears in chemistry research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses MNase-seq in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: MNase-seq is common in secondary-school and first-year university syllabi. It links to neighbouring topics DNA sequencing methods, Molecular biology techniques, so understanding it makes those chapters shorter.
In everyday life: Look for MNase-seq outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study MNase-seq in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what MNase-seq means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain MNase-seq out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is MNase-seq in simple terms?

Why does MNase-seq matter?

Because it connects several chemistry ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study MNase-seq?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on MNase-seq.