DSB Isolation Sequencing Without PCR Bias
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for measuring DNA double-strand breaks (DSBs) in nucleic acids suffer from PCR amplification bias, leading to distorted representations of the original DSB patterns, which is problematic for quantitative genome-wide measurements.
Innovation Solution
A novel DNA library preparation protocol, INDUCE-seq, that avoids PCR amplification before DSB detection, enhancing the signal-to-noise ratio and allowing direct measurement of genomic breaks, where each sequence read corresponds to one DSB.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification is used to enrich DSB sequences before sequencing, then the sensitivity of DSB detection is improved, but PCR amplification bias introduces distortion in the representation of original DSB patterns
Solution Approach 1:
The patent extracts and removes the PCR amplification step from the DSB detection workflow. By using a PCR-free library preparation protocol, the method eliminates the source of amplification bias while maintaining sufficient sensitivity through direct sequencing of DSB-enriched libraries prepared without PCR amplification.
Solution Approach 2:
The patent changes the key parameter of library preparation by transitioning from PCR-based amplification to a PCR-free approach. This parameter change eliminates the nonlinear amplification process that causes bias, allowing linear representation of original DSB frequencies while maintaining detection sensitivity through optimized library construction protocols.
2Productivity
If standard DNA library preparation with PCR amplification is used, then sufficient sequencing depth is achieved, but quantitative measurement of DSB frequency and position becomes impossible
Solution Approach 1:
The patent removes the PCR amplification step that prevents quantitative measurement. By using PCR-free library preparation, each sequencing read directly represents one original DSB molecule, enabling accurate quantification of DSB frequency and position without the distorting effects of amplification bias.
Solution Approach 2:
The patent creates a direct linear copy relationship where each sequencing read is a faithful representation of one original DSB. Without PCR amplification, the sequencing process generates copies that maintain the original frequency and position information, enabling precise quantitative measurement of the DSB landscape.
3Measurement precision
If PCR amplification is performed before sequencing, then low-abundance DSB signals are enhanced, but high levels of noise are introduced into the system
Solution Approach 1:
The patent extracts and eliminates the PCR amplification step that introduces noise. By using PCR-free library preparation, the method avoids amplification artifacts and biases while maintaining sensitivity through direct sequencing, thereby preserving a high signal-to-noise ratio for detecting low-abundance DSBs.
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 both low-level sporadic and high-level recurrent DSBs, providing accurate characterization of their origins and mechanisms of repair, suitable for safe genome-editing development.
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
Figure 1
Figure 2a~2c
Figure 3a
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 aforesaid method including at least a plurality of oligonucleotides for ligating to said 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 said kit and said method.