Multiplex Amplification Detection Assay for Low-Target DNA
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for quantifying nucleic acids, especially in low-target samples, face challenges such as low amounts and fragmentation of DNA, leading to difficulties in detecting multiple targets without risking accuracy due to sample splitting.
Innovation Solution
A method involving targeted pre-amplification of bisulfite-treated DNA using the same primer pairs for both pre-amplification and subsequent PCR-flap assays, allowing for multiplexed analysis without the need for whole-genome pre-amplification or nested primers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If sample splitting is used to test for multiple targets, then testing coverage is improved, but accuracy deteriorates due to false negative results from low copy number targets
Solution Approach 1:
The patent performs pre-amplification of all target sequences before sample splitting and individual target analysis. This preliminary action increases the copy number of all targets uniformly, ensuring that even low-abundance targets have sufficient copies in each aliquot for accurate detection, thereby resolving the contradiction between testing coverage and accuracy
2Productivity
If pre-amplification PCR is carried out with high primer concentration to increase target enrichment, then amplification efficiency is improved, but non-specific background amplification increases
Solution Approach 1:
The patent employs a two-stage amplification strategy with different primer concentrations. The first stage uses high primer concentration for limited cycles to rapidly enrich targets, then switches to low primer concentration for subsequent cycles to maintain specificity. This periodic change in amplification conditions resolves the contradiction between efficiency and background amplification
Solution Approach 2:
The patent dynamically adjusts primer concentration as a parameter during the amplification process. By changing the primer concentration from high to low after an initial enrichment phase, the system optimizes both amplification efficiency and specificity, resolving the contradiction between productivity and harmful background amplification
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 the individual detection of multiple low-copy number targets with reduced risk of false negative results, improving the analysis of nucleic acids in low-target samples.
Implementation Method 1
variants of the polymerase chain reaction (PCR) have become the most powerful and widespread technology
Implementation Method 2
bisulfite treatment is typically used to convert unmethylated cytosine residues to uracil residues
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.