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Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP
Published on: January 20, 2016
Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin
Biochemistry
|December 3, 1985
Summary
Researchers developed a novel method to isolate specific eukaryotic genes as oligonucleosome fragments using DNA hybridization and chromatography. This technique efficiently enriches specific genes for studying gene regulation proteins.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Studying eukaryotic gene regulation requires isolating specific genes within their native chromatin structure.
- Existing methods may not effectively preserve the integrity of chromatin fragments for detailed analysis.
Purpose of the Study:
- To introduce a new technique for isolating specific eukaryotic genes as native oligonucleosome fragments.
- To assess the efficiency and purity of gene isolation using this novel method.
Main Methods:
- Hybridization of single-stranded termini of chromatin restriction fragments to complementary mercurated DNA probes.
- Isolation of DNA-protein hybrids via sulfhydryl-Sepharose chromatography.
- Testing the technique using SV40 minichromosomes and background chromatin from sea urchin embryos.
Main Results:
- Approximately 80% of treated SV40 minichromosomes hybridized to DNA probes under mild conditions.
- SV40 chromatin was reisolated with 88% purity and 63% yield from a mixed chromatin sample.
- This resulted in a 115-fold enrichment of specific genes as chromatin fragments.
- Electron microscopy and gel electrophoresis confirmed the integrity of isolated chromatin proteins.
Conclusions:
- The described technique effectively isolates specific eukaryotic genes as native oligonucleosome fragments.
- This method allows for the structural and biochemical study of proteins involved in gene regulation.
- It is now possible to isolate repeated genes from higher eukaryotes for in-depth analysis.
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