Nuclease Hypersensitive Site Analysis via Aqueous Adapter Ligation
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Solution Overview
Problem
Current methods for mapping multiple hypersensitive sites across a mammalian genome are inefficient, high-cost, and labor-intensive, often resulting in high background noise and prolonged processing times due to the use of low melting agarose gels, which slow down reaction kinetics and complicate automation.
Innovation Solution
A method involving the use of sequence-specific restriction enzymes to fragment chromatin, followed by adapter ligation and secondary digestion in an aqueous medium, allowing for the generation of uniform-sized DNA fragments that can be analyzed without stabilizing process intermediates in gels, thereby reducing background noise and processing time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If low melting agarose gels are used to stabilize process intermediates, then the reliability of the mapping process is improved, but the processing time increases and automation becomes more difficult
Solution Approach 1:
The patent removes the low melting agarose gel stabilization step from the workflow entirely. Process intermediates are handled directly in aqueous solution without gel embedding, eliminating the time-consuming gel preparation, incubation, and removal steps while maintaining reliability through optimized enzyme conditions and buffer compositions.
Solution Approach 2:
The patent replaces the mechanical gel-based stabilization system with a chemical/biochemical solution approach. Instead of using physical gel matrices to stabilize intermediates, the invention uses carefully controlled aqueous buffers with optimized pH, ionic strength, and protective agents to maintain intermediate stability during processing.
2Stability of the object's composition
If low melting agarose gels are used for processing, then the stability of intermediates is improved, but the reaction kinetics slow down
Solution Approach 1:
The patent substitutes the physical constraint of gel matrices with optimized biochemical conditions. DNA fragments are processed in aqueous buffers containing protective agents and optimized ionic conditions that stabilize nucleic acids without impeding molecular diffusion and enzymatic reaction rates.
Solution Approach 2:
The patent optimizes multiple parameters of the aqueous processing medium including pH, temperature, ionic strength, and addition of protective chemicals to simultaneously achieve both DNA fragment stability and rapid reaction kinetics. These parameter optimizations allow enzymes to function at full speed while intermediates remain stable.
3Measurement precision
If traditional methods with multiple processing steps are used, then the precision of hypersensitive site mapping is improved, but the complexity and cost increase
Solution Approach 1:
The patent combines multiple separate processing steps into a streamlined aqueous-based workflow. Adapter ligation, fragment processing, and preparation for analysis are merged into continuous aqueous phase operations, reducing the number of discrete steps while maintaining mapping precision through optimized reaction conditions.
Solution Approach 2:
The aqueous medium serves multiple functions simultaneously: it stabilizes DNA fragments, enables enzyme reactions, allows adapter ligation, and facilitates subsequent analysis preparation. This multi-functional approach replaces multiple specialized reagents and steps required in traditional gel-based methods.
4Reliability
If traditional gel-based methods are used, then the reliability of intermediate handling is improved, but the ease of automation decreases
Solution Approach 1:
The patent extracts the DNA intermediates from the gel matrix environment and processes them entirely in aqueous solution. This eliminates the manual gel manipulation steps that are difficult to automate, allowing liquid handling robots and automated workstations to process samples consistently and reliably.
Solution Approach 2:
The patent replaces manual gel-based handling mechanisms with automated liquid handling systems. The aqueous-based workflow is compatible with robotic pipetting, automated incubation, and high-throughput processing equipment, enabling full automation while maintaining handling reliability through precise programmable control.
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
This approach enables rapid, high-throughput, and cost-effective mapping of hypersensitive sites, facilitating genome-wide analysis without the need for gel stabilization, thus speeding up sample processing and reducing noise, making it suitable for automation and parallel processing of multiple samples.
Implementation Method 1
fragmenting a nucleic acid sample comprising chromatin (e.g. genomic DNA) at multiple hypersensitive sites by treating the nucleic acid sample comprising chromatin with a first sequence specific restriction enzyme
Implementation Method 2
ligating an adapter oligonucleotide onto the sticky end produced by the first sequence specific restriction enzyme
Implementation Method 3
treating the fragments with said second sequence specific restriction enzyme which second sequence specific restriction enzyme cuts at a position at a defined number of bases distal to said recognition site
Data Source
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Figure 2A~2B
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AI summary
The present invention provides a method for analysing nuclease hypersensitive sites which method comprises: i) cleaving a nucleic acid sample comprising chromatin at multiple nuclease hypersensitive sites with a first sequence specific restriction enzyme to introduces a staggered cut and leave a single chain 3' or 5' overhang in a double stranded DNA; ii) optionally isolating substantially free DNA from the digested nucleic acid sample or removing the protein and RNA components from the digested nucleic acid sample to leave substantially free DNA; iii) ligating an adapter oligonucleotide onto the overhang produced by the first sequence specific restriction enzyme in aqueous solution, which adaptor oligonucleotide contains a single stranded region which is complementary to the overhang produced by the first sequence specific restriction enzyme, and which adaptor oligonucleotide contains a recognition site (e.g. target DNA sequence) for a second restriction enzyme; iv) treating the ligated DNA sequence with a second restriction enzyme wherein said second restriction enzyme is specific to said recognition site introduced within said adaptor oligonucleotide, wherein said second restriction enzyme cuts at a position at a defined number of bases distal to said recognition site and introduces a staggered cut, leaving a single chain 3' or 5' overhang in the double stranded DNA; v) optionally amplifying the DNA fragments; vi) analysing the DNA fragments formed in iv) or v) from a plurality of sequences (such as a plurality of genes) wherein at least steps iii) and iv) of the method are conducted in an aqueous medium.