Polymerase-Free Enzyme Mix for DNA Fragmentation
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Solution Overview
Problem
Current DNA fragmentation methods for genome sequencing face challenges such as sequence bias, loss of DNA modifications, and inefficiencies when dealing with low-quality or FFPE DNA samples, particularly due to the introduction of sequence errors and damage during enzymatic fragmentation.
Innovation Solution
A polymerase-free enzyme mix, FRAG, comprising a double-stranded DNA nickase, a single-strand nuclease, and optionally a DNA ligase, is used to fragment DNA without introducing sequence errors, preserving nucleotide modifications and allowing for controlled fragment sizes, suitable for high-throughput sequencing of FFPE samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If enzymatic fragmentation methods utilizing polymerase for nick translation or extension are used, then DNA can be fragmented, but nucleotide modification marks are removed and sequence errors are introduced
Solution Approach 1:
The patent removes the DNA polymerase component from the fragmentation enzyme mix, retaining only the nickase and single-strand nuclease. This extraction of the harmful element (polymerase) eliminates its ability to remove nucleotide modifications and introduce sequence errors, while preserving the desired fragmentation function through the remaining enzymes.
Solution Approach 2:
The patent changes the enzymatic parameters by selecting specific enzymes with particular properties: a nickase that creates nicks without extensive processing, and a single-strand nuclease that cleanly removes overhangs. This parameter selection optimizes the fragmentation process to preserve modifications while achieving adequate fragmentation.
2Productivity
If mechanical shearing methods are used, then DNA can be fragmented, but significant sample loss occurs through shredding and adherence to surfaces
Solution Approach 1:
The patent replaces the mechanical shearing system with an enzymatic system. Instead of using physical forces (acoustic, hydrodynamic, or nebulization) that cause sample loss through shredding and surface adherence, the patent employs biochemical enzymes (nickase and single-strand nuclease) to fragment DNA in solution, eliminating the mechanical losses entirely.
3Ease of manufacture
If existing enzymatic fragmentation methods are used, then cost-effective shearing of large numbers of samples is achieved, but sequence errors and loss of nucleotide modification marks occur
Solution Approach 1:
The patent extracts the problematic DNA polymerase from the enzymatic mix while retaining the cost-effective nature of enzymatic processing. By using only nickase and single-strand nuclease, the method maintains simplicity and scalability for high-throughput applications while eliminating the source of sequence errors and modification loss.
Solution Approach 2:
The patent optimizes enzymatic parameters by selecting specific enzymes and conditions that preserve modifications. The use of a nickase followed by a single-strand nuclease under controlled conditions provides a cost-effective high-throughput solution that also maintains sequence accuracy and modification integrity.
4Productivity
If fragmentation methods are used on FFPE DNA samples, then genomic DNA can be analyzed, but DNA is further degraded or sequence errors are introduced
Solution Approach 1:
The patent replaces mechanical shearing with a gentle enzymatic process that is less harsh on already damaged FFPE DNA. The nickase and single-strand nuclease work under mild conditions to fragment the DNA without causing additional mechanical stress or degradation, preserving the integrity of already compromised samples.
Solution Approach 2:
The patent uses optimized enzymatic parameters (specific enzymes, temperatures, and incubation times) that are gentle on FFPE DNA. The nickase creates nicks without extensive processing, and the single-strand nuclease cleanly removes overhangs without degrading the DNA further, maintaining reliability for sequencing applications.
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
FRAG effectively fragments DNA while preserving nucleotide modifications, reducing sequence bias and artifacts, and improving sequencing metrics, particularly for FFPE samples, by avoiding the use of DNA polymerases and enabling high-quality sequence data from reduced DNA amounts.
Implementation Method 1
a double-stranded DNA nickase preferably one that randomly nicks DNA
Implementation Method 2
a single-strand nuclease
Implementation Method 3
a DNA ligase capable of sealing a nick within a DNA
Data Source
AI summary
Provided herein is a polymerase-free enzyme mix (FRAG) for fragmenting double-stranded DNA. In some embodiments the enzyme mix may comprise a double-stranded DNA nickase and at least one of a DNA ligase capable of sealing a nick within a DNA, and a single-strand specific DNA nuclease. Methods for fragmenting double-stranded DNA are also provided.