Micrococcal Nuclease Digestion
Micrococcal Nuclease Digestion - Related Videos
11:42
Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells
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Cited by 6 •
2019
Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.
12:04
Mouse Genome Engineering Using Designer Nucleases
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Cited by 14 •
2014
Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.
04:57
Chromatin Shearing: A Method to Generate Chromatin Fragments Using Enzymatic Digestion
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2023
This video describes the protocol for chromatin shearing using micrococcal nuclease enzymatic digestion. Chromatin shearing is an essential step in Chromatin immunoprecipitation or ChIp workflow, which helps investigate protein-DNA interaction in human chronic myeloid leukemia cells. In the feature protocol chromatin shearing is performed using sonication.
09:45
Restriction Enzyme Digests
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2023
Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence. These enzymes occur naturally in bacteria as a defense against bacteriophages - viruses that infect bacteria. Bacterial restriction enzymes cut the invading bacteriophage DNA while leaving the bacterial genomic DNA unharmed due to addition of methyl groups. This video explains the basic principles of restriction enzymes including: how restriction enzymes are named and the types of recognition sites and...
07:42
Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)
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Cited by 21 •
2016
Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.
FAQs
Calcium enables micrococcal nuclease activity, so its availability directly affects whether exposed nucleic acid is cut. If calcium conditions are not maintained, digestion may be incomplete or difficult to interpret. Researchers therefore treat calcium availability as a critical reaction variable when comparing samples or using fragment patterns to evaluate chromatin organization.
Histones shield the DNA associated with nucleosomes from nuclease cleavage, whereas linker DNA remains more accessible. The resulting fragments therefore contain information about which nucleic acid regions were protected and which were exposed. This protection pattern allows investigators to distinguish aspects of chromatin organization from general nucleic acid breakdown.
Enzyme concentration, calcium availability, and reaction time are central variables because each can alter the extent of cleavage. Excessive or insufficient digestion can change the fragment pattern and reduce its interpretability. Careful control of these conditions is especially important when the goal is to compare chromatin accessibility or infer nucleosome positioning between samples.
A typical workflow applies micrococcal nuclease to a biological sample under controlled calcium conditions, allows digestion for a defined reaction time, and then examines the nucleic acid fragments produced. The experiment must preserve a consistent enzyme concentration and timing across samples. Researchers interpret the resulting fragments in relation to protected and accessible regions.
Fragment patterns can help estimate nucleosome positioning and reveal differences in chromatin accessibility. They also provide evidence about how DNA interacts with proteins, because protected regions resist cleavage more effectively than exposed regions. These outcomes make the method useful for connecting nucleic acid organization with the physical arrangement of chromatin.
Researchers apply the method when they need to investigate genome organization, DNA-protein interactions, or chromatin features associated with gene regulation. It can help compare how accessible nucleic acid regions are under different biological conditions. In this context, cleavage-derived fragments serve as experimental evidence for changes in chromatin structure rather than as a direct measure of gene activity.