Linked Primer Set for Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current methods for amplifying and detecting multiple target nucleic acids on a solid phase have low identification performance, especially when analyzing a large number of targets or when individual amplification products are difficult to separate, leading to challenges in distinguishing between different nucleic acid samples.
Innovation Solution
A primer set is developed that includes terminal primers and double-headed primers designed to produce linked amplification products, allowing for easier detection by incorporating labeling and binding tags that can bind to a solid-phase support, enabling the use of these tags as indices for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple target nucleic acids are amplified and detected individually on a solid phase, then detection specificity is enhanced, but identification performance deteriorates when analyzing a large number of targets or when individual amplification products are difficult to separate
Solution Approach 1:
The patent combines multiple target nucleic acid sequences into a single linked amplification product. The primer set includes a first primer hybridizing to the first target nucleic acid, a second primer hybridizing to the second target nucleic acid, and a third primer hybridizing to the fourth target nucleic acid, which are designed to amplify and link multiple targets into one continuous product that can be detected as a unified entity on the solid phase.
Solution Approach 2:
The linked amplification product is segmented into distinct regions corresponding to each target nucleic acid sequence. The product comprises a first region from the first target nucleic acid, a second region from the second target nucleic acid, and a third region from the fourth target nucleic acid, allowing each target to be individually identified within the unified product through its specific sequence characteristics.
2Productivity
If multiple target nucleic acids are amplified simultaneously, then detection efficiency is improved, but the ability to distinguish between different nucleic acid samples deteriorates
Solution Approach 1:
Each target nucleic acid region within the linked amplification product possesses unique local sequence characteristics. The first region contains sequences specific to the first target nucleic acid, the second region contains sequences specific to the second target nucleic acid, and the third region contains sequences specific to the fourth target nucleic acid, enabling differentiation of samples based on the presence or absence of specific regional sequences.
3Measurement precision
If individual amplification products are separated for detection, then identification accuracy is improved, but the complexity of the detection system increases
Solution Approach 1:
The detection system captures the entire linked amplification product as a single entity on the solid phase, eliminating the need for complex separation systems. The product is immobilized and detected in its entirety, simplifying the detection apparatus while maintaining the ability to identify multiple targets through their integrated sequence information.
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
The primer set enhances detection efficiency by producing linked amplification products that can be easily identified on a solid-phase support, reducing false positivity and improving the ability to detect multiple target nucleic acids simultaneously with high accuracy.
Implementation Method 1
terminal primer A comprising a polynucleotide comprising, in its 3'-terminal part, a nucleotide sequence that hybridizes to a 3'-terminal part of a complementary sequence of the nucleotide sequence of a first target nucleic acid
Implementation Method 2
a primer set for preparing a nucleic acid amplification product detectable on a solid-phase support
Implementation Method 3
one of the terminal primer A and the terminal primer B further comprises a labeling part which is a labeling agent or is a tag capable of binding to a labeling agent
Implementation Method 4
the other terminal primer further comprises a binding part which is a tag capable of binding to the solid-phase support
Data Source
AI summary
A primer set includes a terminal primer A including, in its 3′-terminal part, a nucleotide sequence that hybridizes to a 3′-terminal part of a complementary sequence of a first target nucleic acid sequence, a k-th double-headed primer including two polynucleotides linked at their 5′ terminal sides, wherein one of the two polynucleotides includes, in its 3′-terminal part, a nucleotide sequence that hybridizes to a 3′-terminal part of a nucleotide sequence of a k-th target nucleic acid, and the other polynucleotide includes, in its 3′-terminal part, a nucleotide sequence that hybridizes to a 3′-terminal part of a complementary sequence of a (k+1)th target nucleic acid, and a terminal primer B including, in its 3′-terminal part, a nucleotide sequence that hybridizes to a 3′-terminal part of a nucleotide sequence of a N-th target nucleic acid.