3C Library Production Using Linker-Preserving Nucleosome Digestion
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Solution Overview
Problem
Existing chromosome conformation capture (3C) methods lack the sensitivity and resolution needed to study interactions between regulatory elements in mammalian genomes, particularly at single-base pair resolution, which is crucial for understanding gene regulation and identifying novel sequences controlling gene expression.
Innovation Solution
A novel process involving cell fixation, permeabilization, and digestion using micrococcal nuclease to produce mono-nucleosomes with intact inter-nucleosomal linkers, followed by ligation and de-crosslinking to create a 3C library, allowing for high-resolution analysis of nucleic acid interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional 3C methods use homogenization to break open cells and release chromatin, then cell lysis is achieved, but resolution is limited and chromatin structure may be disrupted
Solution Approach 1:
The patent replaces the mechanical homogenization system with a biochemical system using micrococcal nuclease enzyme digestion. Instead of mechanically breaking open cells and chromatin, the enzyme specifically digests chromatin in a controlled manner to produce mono-nucleosomes with intact linkers, achieving superior resolution without mechanical disruption.
Solution Approach 2:
The patent changes the digestion parameters by using micrococcal nuclease under controlled conditions to produce mono-nucleosomes of 180-200bp with intact inter-nucleosomal linkers. This precise control of digestion parameters enables single-base pair resolution, dramatically improving measurement precision compared to conventional methods.
2Measurement precision
If restriction enzymes are used for chromatin digestion, then fragmentation is achieved, but resolution is limited to approximately 256 bp due to enzyme recognition site spacing
Solution Approach 1:
The patent changes the fundamental parameter of fragment size by using micrococcal nuclease to produce mono-nucleosomes of 180-200bp with intact linkers, rather than the 256 bp limit imposed by restriction enzyme recognition sites. This parameter change enables single-base pair resolution through ligation of adjacent nucleosomes.
Solution Approach 2:
The patent extracts and utilizes the inter-nucleosomal linker regions that connect adjacent nucleosomes. By preserving these linkers during digestion and enabling their ligation, the method captures chromatin conformation information at a resolution of single base pairs, overcoming the resolution limit of restriction enzymes.
3Measurement precision
If DNaseI or Micrococcal nuclease are used for digestion, then chromatin fragmentation is achieved, but previous enrichment steps did not result in higher resolution data in larger mammalian genomes
Solution Approach 1:
The patent performs preliminary action by specifically digesting chromatin to mono-nucleosomes with intact linkers before ligation. This preliminary digestion step, using micrococcal nuclease under controlled conditions, prepares the chromatin in an optimal state for subsequent ligation, enabling high-resolution data generation in mammalian genomes.
Solution Approach 2:
The patent creates a composite structure by ligating mono-nucleosomes with their intact inter-nucleosomal linkers. This composite approach combines the precision of nucleosome positioning with the conformational information preserved in the linkers, achieving superior resolution and data quality in mammalian genome studies.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Achieves single-base pair resolution, enabling detailed study of regulatory elements and their interactions, facilitating the identification of novel sequences controlling gene expression and improving the accuracy of genome-wide association studies for personalized medicine and diagnostics.
Implementation Method 1
cross-linking the chromatin within the cells in a population of eukaryotic cells
Implementation Method 2
fragmenting the cross-linked chromatin to produce nucleic acid fragments; wherein the chromatin is fragmented to produce mono-nucleosomes of 180-200bp with the inter-nucleosomal linkers attached
Implementation Method 3
ligating the mono-nucleosomes of 180-200bp with the inter-nucleosomal linkers attached to produce ligated nucleic acid fragments
Data Source
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AI summary
The present invention relates to a process for producing a chromatin conformation capture (3C) library. This may be used for identifying nucleic acid regions within a nucleic acid sample which interact with one another. The process comprises treating nucleic acids in a population of eukaryotic cells, the process comprising the steps: (i) immobilising the nucleic acids within the cells in a population of eukaryotic cells; (ii) permeabilising or removing the cell membranes of the eukaryotic cells; and (iii) fragmenting the immobilised nucleic acids within the cells to produce nucleic acid fragments.