Short-Chain Nucleic Acid Elongation Primer Set for LAMP Detection
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Solution Overview
Problem
Current methods face challenges in detecting and amplifying short-chain nucleic acids, such as siRNA and miRNA, due to their short sequence lengths, which complicates primer annealing, amplification, and sensitivity, hindering their clinical use as diagnostic tools.
Innovation Solution
A short-chain nucleic acid elongation primer set is developed, comprising first and second elongation primers that hybridize with specific sequences to produce an elongated, complementary double-stranded product, facilitating detection and amplification through a loop-mediated isothermal amplification method.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (Northern blotting, microarray analysis, real-time PCR) are used for short-chain nucleic acids, then detection can be performed, but the short sequence length makes it difficult to ensure primer annealing region, leading to poor amplification and detection sensitivity
Solution Approach 1:
The invention divides the detection process into two distinct stages: (1) elongation stage using elongation primers to extend the short-chain nucleic acid into a longer intermediate product, and (2) amplification stage using LAMP primers to amplify the elongated product. This segmentation allows each primer set to be optimized for its specific function, resolving the primer design complexity while improving detection sensitivity.
Solution Approach 2:
The invention performs preliminary elongation of the short-chain nucleic acid before amplification. By first extending the short template into a longer intermediate product with sufficient annealing regions, the subsequent LAMP amplification can proceed efficiently. This preliminary action solves the fundamental problem of insufficient primer binding sites in short-chain nucleic acids.
2Productivity
If standard PCR amplification is attempted with short-chain nucleic acids, then amplification may occur, but nonspecific amplification increases and detection accuracy decreases
Solution Approach 1:
The invention introduces an intermediate elongated product as a mediator between the short-chain nucleic acid template and the LAMP amplification. This intermediate product serves as a reliable template with sufficient length and structure for specific LAMP primer binding, thereby maintaining high amplification efficiency while ensuring amplification specificity and reducing nonspecific products.
3Measurement precision
If the length of nucleic acid is increased through elongation, then detection sensitivity improves, but the complexity of the amplification system increases
Solution Approach 1:
The invention uses Phi29 DNA polymerase which possesses multiple functions: it can perform both the elongation of short-chain nucleic acids and the subsequent LAMP amplification. This multi-functionality reduces the need for multiple specialized enzymes and complex system setup, thereby improving detection sensitivity while minimizing the increase in system complexity.
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 specific and efficient elongation and amplification of short-chain nucleic acids, improving detection sensitivity and accuracy, even at low concentrations, and reducing nonspecific amplification, thus enhancing their utility as diagnostic tools.
Implementation Method 1
a first elongation primer sequence which hybridizes with the first sequence
Implementation Method 2
elongating the first sequence to obtain an elongated intermediate product containing a complementary sequence of the first sequence
Implementation Method 3
a second elongation primer sequence which hybridizes with the elongated intermediate product
Implementation Method 4
elongating the second elongation primer and the elongated intermediate product to obtain an elongated product
Implementation Method 5
One of the third sequence, the complementary sequence of the 1′-th sequence, and the fifth sequence is a loop primer recognition sequence
Data Source
AI summary
According to one embodiment, a primer set for elongating a target short-chain nucleic acid containing a first sequence to obtain an elongated product is provided. The elongated product contains a second, a third, a fourth sequence, a complementary sequence of the 1′-th sequence and a sixth sequence. The complementary sequence of the 1′-th sequence is a loop primer sequence. The primer set contains a first elongation primer containing a first elongation primer sequence and a complementary sequence of the sixth sequence, and a second elongation primer containing a second elongation primer sequence, the fourth, the third, and the second sequence.