Isothermal Pre-Amplification for Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid amplification techniques face challenges in performing multiplexed assays, where multiple target sequences are simultaneously amplified and detected in the same reaction mixture, due to variations in primer stability, structural differences, and competition among targets, leading to variable and erroneous signal yields.
Innovation Solution
A method involving a target probe and a detection probe, where the target probe is modified by adding nucleotide bases in the presence of a polymerase, and then ligated with the detection probe to form a two-probe ligation product, which is detected, allowing for the amplification and analysis of multiple target sequences in a single reaction vessel under isothermal conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional nucleic acid amplification methods are used for multiplexed assays, then multiple target sequences can be simultaneously amplified, but variable and erroneous signal yields occur due to primer stability differences and competition among targets
Solution Approach 1:
The assay is divided into two distinct phases: an isothermal pre-amplification phase that generates intermediate products, and a subsequent PCR detection phase. This segmentation allows the pre-amplification to occur under conditions that minimize competition effects, while the PCR phase provides robust detection, thereby resolving the contradiction between multiplexing capability and signal consistency.
Solution Approach 2:
The invention performs preliminary isothermal pre-amplification of all target sequences before the main PCR detection. This preliminary action equalizes the starting amounts of different targets, reducing the impact of primer stability differences and competition effects during the subsequent PCR, thus improving signal yield consistency while maintaining multiplexing capability.
2Adaptability or versatility
If multiple primers are used for different target sequences, then multiplexed detection is enabled, but competition among primers leads to variable amplification efficiency
Solution Approach 1:
The invention changes the reaction parameters by using isothermal conditions for pre-amplification instead of thermal cycling. This parameter change reduces the competitive interactions between multiple primers that occur during conventional PCR, leading to more uniform amplification efficiency across multiple targets while maintaining the ability to detect multiple analytes simultaneously.
3Measurement precision
If conventional PCR amplification is used, then high sensitivity detection is achieved, but thermal cycling causes variable primer renaturation rates among different targets
Solution Approach 1:
The invention performs preliminary isothermal pre-amplification before the main PCR detection. This preliminary action occurs under controlled isothermal conditions that ensure consistent primer hybridization across all targets, eliminating the variability caused by thermal cycling. The subsequent PCR then provides high-sensitivity detection of the pre-amplified products.
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 method enables consistent and quantitative amplification and detection of target nucleic acid sequences, improving multiplex capabilities by reducing competition and variability among targets, and allowing for flexible and sensitive detection of multiple analytes.
Implementation Method 1
mixing the sample with a target probe having a sequence capable of hybridizing with the target sequence, under conditions effective to form a double-stranded complex of the analyte and the probe
Implementation Method 2
reacting the probe in the complex in the presence of a polymerase and a selected one to three of four possible nucleotide triphosphates, thereby to add a selected one or more target-directed nucleotide bases to the probe's 3' end to produce a modified probe
Implementation Method 3
by the reacting ligating the modified probe with the detection probe to form a two-probe ligation product
Data Source
AI summary
A method and kit for detecting the presence of a target sequence in a polynucleotide analyte contained in a sample are disclosed. In practicing the method, the sample is mixed with a single-stranded DNA target probe having a sequence capable of hybridizing with the target sequence, under conditions effective to form a double-stranded complex of the analyte and the single-stranded DNA target probe, and the single-stranded DNA target probe in the complex is reacted in the presence of a polymerase and one to three nucleotide triphosphates, to add a selected one or more target-directed nucleotide bases to single-stranded DNA target probe's 3′ end to produce a modified probe. The modified probe is hybridized with a single-stranded DNA detection probe, the two probes are ligated to form a two-probe ligation product, and the presence of the ligation product is detected.