DNA Double-Strand Break Isolation Using PCR-Free Direct Sequencing
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Solution Overview
Problem
Existing methods for measuring DNA double-strand breaks (DSBs) in nucleic acids, particularly through next-generation sequencing (NGS), suffer from PCR amplification bias, leading to distorted representations of the original DSB patterns and making quantification impossible.
Innovation Solution
A novel DNA library preparation protocol, INDUCE-seq, that avoids PCR amplification by directly measuring DSBs through a method involving oligonucleotide ligation and sequencing, enhancing the signal-to-noise ratio and allowing direct measurement of genomic breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification is used in DNA library preparation for NGS, then the sensitivity and detection capability of DSBs is improved, but PCR amplification bias distorts the original DSB patterns and makes quantification impossible
Solution Approach 1:
The invention extracts and removes the PCR amplification step from the DNA library preparation workflow. By using a PCR-free protocol where adaptors are directly ligated to fragmented DNA and sequenced, the method eliminates the source of amplification bias while maintaining sufficient sensitivity through direct sequencing of the DNA fragments containing DSB repair signatures.
Solution Approach 2:
The invention changes the key parameter of library preparation from PCR-based amplification to PCR-free direct sequencing. This parameter change fundamentally alters the detection mechanism, allowing direct measurement of DSB repair outcomes without the intermediate amplification step that causes distortion of the original DSB pattern information.
2Quantity of substance
If standard DNA library preparation with PCR amplification is used, then the quantity of DNA sufficient for sequencing is achieved, but the quantification of original DSB composition becomes impossible due to amplification bias
Solution Approach 1:
The invention removes the PCR amplification step that creates the conflict between sufficient DNA quantity and accurate DSB quantification. By using direct ligation and sequencing methods, the protocol generates sufficient library material for sequencing while preserving the authentic abundance information of different DSB repair outcomes.
Solution Approach 2:
Instead of using PCR to create copies of DNA fragments (which introduces bias), the invention uses direct sequencing methods that create digital copies through sequencing reads. Each read represents an authentic copy of the original DNA fragment, preserving the true relative abundances of different DSB repair signatures without amplification distortion.
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
INDUCE-seq simultaneously detects low-level sporadic and high-level recurrent DSBs, providing a direct and accurate measurement of DSBs without PCR bias, suitable for understanding DSB formation and repair mechanisms.
Implementation Method 1
exposing a sample of nucleic acid suspected of containing DSBs, under ligation conditions, to a first pair of oligonucleotides a first one of which comprises a 5′ binding feature that enables ligation of said oligonucleotide to a first strand of said DSB
Data Source
AI summary
The invention relates to a method for determining the number and nature of DNA double-strand breaks (DSBs) in a nucleic acid sample, ideally gDNA; a kit of parts for performing the method including at least a plurality of oligonucleotides for ligating to the nucleic acid sample and providing at least a first hybridization site (RD1 SP or RD2 SP) to which at least one read sequencing primer can hybridise; and oligonucleotides for use in the kit and method.