Two-Tailed RT Primers for Multiplex Short Nucleic Acid Detection

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Solution Overview

Problem

Current methods for amplifying and detecting short nucleic acid biomarkers in liquid biopsies, such as miRNA, face challenges due to their rarity and the difficulty in achieving specific amplification and detection, particularly in multiplex assays where multiple biomarkers need to be monitored simultaneously.

Innovation Solution

The use of two-tailed RT primers with shared arm, stem, and loop sequences, combined with universal forward PCR primers and modified nucleotide probes, allows for specific multiplex amplification and detection of short nucleic acids like miRNA, reducing non-specific amplification and enhancing probe specificity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If amplification primers are used to increase sensitivity of detection, then detection sensitivity is improved, but specificity is reduced due to shorter complementary sequences

Engineering Contradiction:

Improvedetection sensitivityVSAvoidamplification specificity

Core Design Contradiction:

Measurement precisionVSManufacturing precision

Solution Approach 1:

The primer is divided into functionally distinct segments: a target-complementary region for specific binding and a universal arm-stem-loop structure for amplification. This segmentation allows each region to optimize its function - the complementary region maintains specificity while the universal structure enables sensitive amplification detection

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The arm-stem-loop structure serves multiple functions: it provides a universal binding site for the forward PCR primer, forms a stable secondary structure to enhance primer efficiency, and enables multiplexing capability. This multi-functionality resolves the contradiction by allowing sensitive amplification without compromising target specificity

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Measurement precision

If traditional two-tailed RT primers are used for single target detection, then detection specificity is maintained, but device complexity and assay time increase when detecting multiple biomarkers

Engineering Contradiction:

Improvedetection specificityVSAvoidassay complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The RT primer incorporates a universal arm-stem-loop structure that can be paired with different target-specific complementary sequences to detect multiple biomarkers. This allows a single primer design framework to handle multiple targets, reducing assay complexity while maintaining specificity through the target-specific complementary regions

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention merges the RT primer design with the amplification primer structure by integrating the arm-stem-loop element into the RT primer itself. This consolidation eliminates the need for separate amplification primers and reduces the number of reagents needed for multiplex detection, thereby reducing device complexity

Inventive Principle:

Principle #5Merging (Combining)

3Adaptability or versatility

If multiple primers and probes are used for multiplex detection, then detection versatility is improved, but background signals and non-specific amplification increase

Engineering Contradiction:

Improvemultiplex detection capabilityVSAvoidnon-specific amplification

Core Design Contradiction:

Adaptability or versatilityVSObject-generated harmful factors

Solution Approach 1:

The arm-stem-loop structure creates a localized region of high stability and specific binding characteristics. The stem-forming sequence ensures that only primers with exact complementarity to the arm region will bind efficiently, reducing non-specific amplification while maintaining versatility for detecting multiple targets with different complementary sequences

Inventive Principle:

Principle #3Local quality

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 efficient and sensitive detection of multiple short nucleic acid biomarkers with reduced background signals, improving the sensitivity and dynamic range of multiplex assays compared to traditional methods.

Implementation Method 1

i) a sequence complementary to the 5′ end of a first target nucleic acid

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

a first detectably labeled probe comprising 5′ to 3: i) a sequence complementary to the second stem sequence and/or the second arm sequence; and ii) a sequence complementary to the sequence complementary to the 3′ end of the first target nucleic acid

Methodology Applied

Scientific EffectNucleic acid hybridization:

Data Source

PatentUS11584960B2Multiplex detection of short nucleic acids

Publication Date: 2023.02.21 ROCHE MOLECULAR SYSTEMS INC

AI summary

Provided herein are methods and compositions for performing multiplex RT-PCR to amplify short nucleic acids.