Low-Bias Sequential Multiplex PCR for Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid amplification methods, particularly multiplex PCR, suffer from amplification bias and high background noise, leading to undesirable artifacts and the need for separate detection of each target signal, which complicates analysis of complex nucleic acid mixtures.
Innovation Solution
A method involving preamplification with equal concentrations of primer pairs followed by a multiplex PCR assay using the same primer pairs and labeled probes, allowing for combined signal detection without separate measurement, utilizing a low-bias amplification buffer and FRET cassettes to reduce bias and noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiplex PCR is used to amplify multiple target nucleic acids simultaneously, then detection capability is improved, but amplification bias and background noise increase
Solution Approach 1:
The amplification process is divided into two sequential steps: a preamplification step using first primer pairs to enrich target nucleic acids, followed by a second amplification step using second primer pairs. This segmentation allows each step to be optimized independently, reducing amplification bias while maintaining the ability to detect multiple targets simultaneously.
Solution Approach 2:
A preamplification step is performed before the main amplification to enrich the target nucleic acids. This preliminary action increases the starting material for the second amplification step, improving detection sensitivity while using lower primer concentrations in the final step to minimize background noise and artifacts.
2Adaptability or versatility
If multiple primer pairs are used in multiplex PCR, then detection of multiple targets is enabled, but background noise and artifacts increase
Solution Approach 1:
The primer pairs are divided into two sets used in sequential steps. The first primer pairs are used in preamplification with higher concentrations to enrich targets, while the second primer pairs are used in the final amplification at lower concentrations to minimize non-specific binding and background noise, thereby enabling clean multiplex detection.
Solution Approach 2:
The preamplification step with first primer pairs performs a preliminary enrichment of target nucleic acids before the final amplification. This ensures that when the second primer pairs are used in the main amplification step, the background noise is minimized because the targets are already enriched, allowing for cleaner multiplex detection.
3Measurement precision
If separate detection of each target signal is performed, then measurement precision is improved, but analysis complexity increases
Solution Approach 1:
Multiple target nucleic acids are amplified and detected in a single multiplex PCR reaction using different primer pairs and probes. The signals from all targets are combined and analyzed together, maintaining measurement precision through optimized amplification conditions while simplifying the analysis by eliminating the need for separate detection steps for each target.
4Productivity
If standard PCR conditions are used for preamplification, then amplification efficiency is improved, but non-specific amplification increases
Solution Approach 1:
The amplification is segmented into two steps with different conditions. The preamplification step uses higher primer concentrations and standard PCR conditions to achieve high amplification efficiency and enrich targets. The second amplification step uses lower primer concentrations and optimized conditions to minimize non-specific amplification, thereby maintaining productivity while reducing harmful non-specific products.
Solution Approach 2:
The preamplification step performs a preliminary enrichment of target nucleic acids using standard PCR conditions and higher primer concentrations. This preliminary action achieves high amplification efficiency and target enrichment. The final amplification step then uses reduced primer concentrations to complete the amplification while minimizing non-specific amplification, balancing productivity with specificity.
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 reduces amplification bias and background noise, enabling efficient multiplexed detection of multiple nucleic acids in a single reaction, simplifying analysis and eliminating the need for separate signal measurement.
Implementation Method 1
Methods for detection and quantification of nucleic acids or nucleic acid sequences, variants of the polymerase chain reaction (PCR) have become the most powerful and widespread technology
Implementation Method 2
at least four different target-specific probe oligonucleotides, wherein each target-specific probe oligonucleotide specifically hybridizes to a different one of the amplified regions
Data Source
AI summary
Provided herein is technology relating to multiplex preamplification of multiple different target nucleic acids from a sample, e.g., an environmental or biological sample, and particularly, but not exclusively, to methods, compositions, kits, and related uses for detecting and assessing mixtures of target nucleic acids, e.g.. The technology provides methods, compositions, and kits for using low-bias amplification conditions to perform highly-multiplexed preamplification followed by highly-multiplexed detection assays such as PCR assays, next generation sequencing assays, and PCR-flap assay for assaying samples that comprise multiple different target nucleic acids.