Plasma DNA Isolation and Multiplex Detection for Low-Copy Targets
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods struggle to accurately detect and quantify low-level, fragmented nucleic acids, particularly in samples like plasma, due to inefficiencies in purification and amplification processes, leading to false negatives and loss of target DNA during analysis.
Innovation Solution
A method involving bisulfite-treated DNA pre-amplification using the same primer pairs for both rounds of amplification, combined with a PCR-flap assay, to enhance detection of multiple targets in low-copy samples, utilizing a combination of PCR and FEN-1-mediated flap cleavage for signal amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If routine purification methods are used on fragmented DNA, then purification is achieved, but DNA loss occurs due to poor binding to purification columns or inefficient alcohol precipitation
Solution Approach 1:
The patent changes the physical-chemical parameters of the purification process by using magnetic beads with specific surface properties and optimized binding/washing/elution conditions to improve DNA recovery efficiency while minimizing loss of fragmented DNA
Solution Approach 2:
The patent introduces magnetic beads as an intermediary substance to facilitate DNA purification, replacing traditional column-based methods and improving recovery of fragmented DNA through controlled magnetic separation
2Productivity
If pre-amplification PCR is carried out with high primer concentration to amplify low-copy targets, then amplification efficiency is improved, but non-specific background amplification and artifacts increase
Solution Approach 1:
The patent employs periodic thermal cycling with optimized temperature profiles and staged amplification cycles to enhance specific amplification while suppressing non-specific background, allowing efficient amplification of low-copy targets without artifacts
Solution Approach 2:
The patent performs preliminary optimization of primer concentration and cycle conditions before full amplification, and uses preliminary blocking steps to prevent non-specific binding, ensuring high specificity from the start of the amplification process
3Adaptability or versatility
If sample is divided into multiple aliquots for testing multiple targets, then comprehensive target detection is achieved, but false negative results increase due to low copy numbers in each aliquot
Solution Approach 1:
The patent segments the detection process into separate reaction chambers or spatially separated zones within a single reaction, allowing multiple targets to be detected simultaneously without dividing the limited DNA sample into multiple aliquots
Solution Approach 2:
The patent merges multiple target detection reactions into a single shared amplification reaction by using primer pairs with similar thermal characteristics and optimized concentrations, enabling comprehensive multi-target analysis from limited DNA copies
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 allows for the reliable detection of multiple low-copy nucleic acid targets with reduced false negatives, maintaining the integrity of the DNA and improving the yield of nucleic acid analysis in large plasma samples.
Implementation Method 1
utilizing a combination of PCR and FEN-1-mediated flap cleavage for signal amplification
Data Source
AI summary
Provided herein is technology relating to the amplification-based detection of bisulfite-treated DNAs and particularly, but not exclusively, to methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of the sample DNA. The technology further provides methods for isolating DNA from blood or blood product samples, e.g., plasma samples.