Nucleic Acid Fragmentation via Methylation-Dependent Restriction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current DNA fragmentation methods for sequencing are complex, enzyme-sensitive, and often require multiple steps, leading to issues like non-random fragmentation, enzyme availability limitations, and increased technical complexity, which can result in suboptimal fragment lengths and increased sequencing errors.
Innovation Solution
A method involving amplification of nucleic acid templates using a dNTP mix containing 5-methyl dCTP, followed by digestion with a methylation-dependent restriction endonuclease like MspJI to produce fragments, which are then directly ligated with adaptors, allowing for controlled fragment length and high-fidelity sequencing without additional enzyme treatments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If mechanical fragmentation or dsDNA fragmentase is used, then DNA can be fragmented, but additional steps are required to polish ends and phosphorylate 5' ends, increasing technical complexity
Solution Approach 1:
The invention extracts and removes the problematic additional steps (polishing and phosphorylation) from the fragmentation workflow by using a natural enzyme (restriction endonuclease) that produces fragments with already-ready ends for direct adapter ligation, eliminating the need for separate polishing and phosphorylation steps
Solution Approach 2:
The restriction endonuclease serves multiple functions simultaneously: it fragments the DNA and produces ends that are directly ready for adapter ligation, combining what were previously separate functions into a single enzymatic step
2Productivity
If transposase or dsDNA fragmentase is used, then fragmentation can be achieved, but the reaction is highly sensitive to enzyme to DNA ratio, requiring careful quantification and control
Solution Approach 1:
The invention changes the critical parameter from enzyme to DNA ratio sensitivity to DNA concentration independence by using restriction endonucleases that recognize specific sequences, making the fragmentation process robust to variations in DNA concentration and eliminating the need for precise quantification
3Ease of manufacture
If transposase-mediated fragmentation is used, then DNA can be fragmented and ligated, but the fragmentation is not truly random due to bias for certain insertion sites
Solution Approach 1:
The invention introduces a sequence-specific recognition mechanism as an intermediary step: the restriction endonuclease first recognizes specific DNA sequences, then cleaves at defined positions relative to those sequences, providing a predictable and uniform fragmentation pattern that is more random than transposase while maintaining precision
4Productivity
If enzyme-based fragmentation is used, then DNA can be fragmented, but excess enzyme must be quickly stopped to prevent over-fragmentation, requiring stop buffer addition that can adversely affect downstream steps
Solution Approach 1:
The invention uses a disposable heat inactivation step instead of a chemical stop buffer: the restriction endonuclease is heat-inactivated by heating to 65°C for 20 minutes, which eliminates the need for chemical stop buffers that could interfere with downstream adapter ligation and sequencing steps
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 simplifies the fragmentation process, reduces technical complexity, and enables efficient production of fragments suitable for sequencing, independent of DNA concentration or enzyme amounts, with high fidelity and adaptable fragment lengths for various sequencing applications.
Implementation Method 1
digesting the product nucleic acid molecules with a methylation-dependent restriction endonuclease, e.g., an MspJI family restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosines
Data Source
Figure 1
Figure 2a~2c
Figure 3
AI summary
Providing herein, among other things, is a method for preparing a nucleic acid for sequencing. In some embodiments, the method comprises a) amplifying a nucleic acid template using a dNTP mix that contains 5-methyl dCTP, thereby producing product nucleic acid molecules that contains methylcytosines; b) digesting the product nucleic acid molecules with a methylation-dependent restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosine and producing fragments of the product nucleic acid molecules; and c) ligating double-stranded adaptors onto the ends of the fragments to produce adaptor- ligated products.