Multiphase Nucleic Acid Amplification for Low-Interference Multiplex Detection
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Solution Overview
Problem
Current nucleic acid amplification methods, particularly multiplex reactions, face challenges such as preferential amplification, interference between analytes, and reduced sensitivity due to competing reactions, leading to costly and inefficient assays.
Innovation Solution
The method involves conducting nucleic acid amplification in multiple phases, where the initial phase prevents exponential amplification, followed by a second phase that allows exponential amplification, thereby focusing resources on desired reactions and reducing interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiplex amplification is conducted in a single phase with all reagents present, then amplification of multiple targets can be achieved, but preferential amplification and interference between analytes occur leading to reduced sensitivity
Solution Approach 1:
The amplification process is divided into two distinct phases: a first phase that generates initial amplification products without exponential amplification, and a second phase that enables exponential amplification. This segmentation prevents preferential amplification of certain targets while maintaining multiplex capability, thereby resolving the contradiction between versatility and measurement precision.
Solution Approach 2:
The first phase performs preliminary amplification to generate sufficient template material for all targets before the second phase begins exponential amplification. This preliminary action ensures that all analytes, including those at low concentrations, have adequate template available when exponential amplification starts, preventing interference and improving detection sensitivity.
2Productivity
If exponential amplification is allowed from the start, then amplification efficiency is high, but competing reactions cause interference and reduce precision
Solution Approach 1:
The amplification process is divided into two distinct phases: a first phase that generates initial amplification products without exponential amplification, and a second phase that enables exponential amplification. This segmentation prevents preferential amplification of certain targets while maintaining multiplex capability, thereby resolving the contradiction between versatility and measurement precision.
Solution Approach 2:
The first phase performs preliminary amplification to generate sufficient template material for all targets before the second phase begins exponential amplification. This preliminary action ensures that all analytes, including those at low concentrations, have adequate template available when exponential amplification starts, preventing interference and improving detection sensitivity.
3Ease of operation
If all amplification reagents are present in the reaction mixture at the start, then the reaction can proceed immediately, but resource competition reduces sensitivity for low-abundance targets
Solution Approach 1:
The amplification process is divided into two distinct phases: a first phase that generates initial amplification products without exponential amplification, and a second phase that enables exponential amplification. This segmentation prevents preferential amplification of certain targets while maintaining multiplex capability, thereby resolving the contradiction between versatility and measurement precision.
Solution Approach 2:
The first phase performs preliminary amplification to generate sufficient template material for all targets before the second phase begins exponential amplification. This preliminary action ensures that all analytes, including those at low concentrations, have adequate template available when exponential amplification starts, preventing interference and improving detection sensitivity.
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 enhances sensitivity and precision in nucleic acid detection, allowing for more efficient amplification of multiple targets with reduced interference and lower reagent consumption.
Implementation Method 1
contacting the sample with a first amplification oligonucleotide under conditions allowing hybridization of the first amplification oligonucleotide to a portion of the target nucleic acid sequence
Data Source
AI summary
Improved methods for use in nucleic acid amplification, including multiplex amplification, where the amplification is carried out in two or more distinct phases are disclosed. The first phase amplification reaction preferably lacks one or more components required for exponential amplification. The lacking component is subsequently provided in a second, third or further phase(s) of amplification, resulting in a rapid exponential amplification reaction. The multiphase protocol results in faster and more sensitive detection and lower variability at low analyte concentrations. Compositions for carrying out the claimed methods are also disclosed.