DNA Fragmentation via Nick Translation Enzymatic System
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Solution Overview
Problem
Current DNA fragmentation methods for next-generation sequencing, such as sonication and enzymatic shearing, are sensitive to reaction conditions and enzyme-to-DNA ratios, leading to inconsistent fragment lengths and requiring additional end repair steps, which complicates library preparation and sequencing.
Innovation Solution
A method using a combination of enzymes with nick translating activity, modified deoxyribonucleoside triphosphates, and modification-sensitive nicking endonucleases that prevent further nicking at specific sites, allowing for controlled DNA fragmentation to produce fragments of predetermined sizes ranging from 50 bp to 50 kbp without the need for additional end repair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If sonication is used to shear DNA, then DNA can be fragmented to desired size, but reactive oxygen species are produced that damage DNA and end repair is required
Solution Approach 1:
The patent replaces the mechanical sonication method with an enzymatic system consisting of a nicking endonuclease and a DNA polymerase. The nicking endonuclease introduces nicks at specific sites, and the polymerase extends these nicks to create controlled breaks, eliminating the need for mechanical ultrasonic vibration that generates harmful reactive oxygen species.
Solution Approach 2:
The patent introduces a nicking endonuclease as an intermediary enzyme that specifically recognizes and nicks at particular DNA sequences. This intermediary provides controlled, site-specific fragmentation rather than random shearing, and the subsequent polymerase extension creates clean breaks without damaging the DNA ends, eliminating the need for end repair.
2Manufacturing precision
If enzymatic methods are used to fragment DNA, then fragmentation can be controlled, but the methods are highly sensitive to enzyme-to-DNA ratio and require precise control of multiple parameters
Solution Approach 1:
The patent changes the kinetic parameters of the enzymatic reaction by using a nicking endonuclease with low activity that introduces nicks slowly, followed by a polymerase with high processivity that extends these nicks rapidly. This parameter mismatch creates a unidirectional reaction that is insensitive to enzyme-to-DNA ratio variations, as the slow nicking step becomes the rate-limiting step regardless of polymerase amount.
Solution Approach 2:
The patent performs preliminary action by having the nicking endonuclease create nick sites before the polymerase extends them. This sequential action ensures that fragmentation only occurs at predetermined sites, and the reaction proceeds in a controlled manner that is less sensitive to variations in enzyme concentrations or incubation time.
3Manufacturing precision
If conventional enzymatic fragmentation is used, then DNA can be fragmented, but fragments contain overhangs requiring end repair
Solution Approach 1:
The patent merges two enzymatic functions into a single reaction system: the nicking endonuclease creates nicks at specific sites, and the DNA polymerase simultaneously extends these nicks to create clean, blunt-ended breaks. This combination of nicking and extension in one reaction produces fragments with ready-to-ligate ends, eliminating the need for separate end repair steps.
Solution Approach 2:
The patent ensures continuity of useful action by having the polymerase immediately extend the nicks created by the nicking endonuclease without interruption. This continuous action transforms the nick sites into clean breaks with proper 3' hydroxyl ends, maintaining the useful fragmentation function while simultaneously preparing the ends for ligation without requiring additional repair 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 enables consistent and controlled DNA fragmentation across a wide DNA concentration range, producing blunt-ended fragments suitable for sequencing, reducing the complexity of library preparation and sequencing processes, and allowing for automated processing without the need for specialized equipment.
Implementation Method 1
at least one modification-sensitive nicking endonuclease that is prevented from nicking DNA if the recognition site(s) contain the modified base
Implementation Method 2
one or more polymerases capable of nick translating activity
Implementation Method 3
a dNTP mix comprising a dNTP having a modified base; and at least one modification-sensitive nicking endonuclease that are prevented from nicking DNA if its recognition site contains the modified base
Data Source
AI summary
Providing herein, among other things, are kits, compositions and methods that relate to DNA fragmentation. An embodiment of a composition provides combining: one or more enzymes capable of nick translating activity, a dNTP mix comprising at least one dNTP having a modified base, and at least one modification-sensitive nicking endonuclease that is prevented from nicking DNA if its recognition site contains the modified base. When the composition is added to a sample comprising a double-stranded DNA template that comprises recognition sites for the modification-sensitive nicking endonuclease, a reaction mix was produced which could be incubated for any time period in excess of about 5 minutes to produce fragments of a desired size of the double-stranded DNA template. In this method, the fragments produced include the modified base and, as such, are not re-nicked by the nicking endonuclease.