Stem-Loop Polynucleotide Primer for Specific Gene Amplification
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Solution Overview
Problem
In the medical field, samples often contain small amounts of nucleic acids as mixtures, making it necessary to amplify target nucleic acids for accurate analysis, especially when variations like mutations or gene fusions occur, requiring a technology for specific amplification of disease-specific genes to reduce costs.
Innovation Solution
A polynucleotide with specific regions for complementary binding, identical sequence matching, and self-hybridization to form a stem-loop structure is used to amplify target nucleic acids, serving as a primer for sequencing and amplification, including a first region complementary to the target, a second region identical to the target, and a third region forming a stem-loop structure.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional amplification methods are used on mixed nucleic acid samples, then the amount of target nucleic acid can be increased, but the amplification lacks specificity and amplifies non-target sequences as well
Solution Approach 1:
The polynucleotide is divided into three functional regions: a first region (nucleotides 1-15) complementary to the target sequence for specific binding, a second region (nucleotides 16-30) identical to the target sequence for self-hybridization, and a third region (nucleotides 31-45) forming a stem-loop structure. This segmentation allows each region to perform its specific function, achieving both amplification and high specificity.
Solution Approach 2:
The invention changes the structural parameters of the primer by incorporating a stem-loop structure formed by self-hybridization of the second region. This structural parameter change enables the primer to specifically recognize and bind to the target sequence while preventing non-specific amplification of mixed nucleic acids.
2Measurement precision
If amplification is performed on samples with mutations or gene variations, then disease-specific genes can be detected, but conventional methods cannot specifically distinguish mutated sequences from wild-type sequences
Solution Approach 1:
The first region of the polynucleotide is designed with local quality matching the specific target sequence, including mutated sequences. By adjusting the nucleotide sequence in the first region to match the specific mutation of interest, the primer can specifically bind to and amplify only the desired mutated sequence, achieving high detection accuracy for specific gene variations.
Solution Approach 2:
The second and third regions provide universal functionality through self-hybridization and stem-loop structure formation, which work consistently across different target sequences. This allows the same basic primer structure to be adapted for detecting various gene variations by simply changing the first region's sequence, providing both specificity and versatility.
3Measurement precision
If sequencing is performed on all genes in a sample, then comprehensive analysis is achieved, but the cost increases significantly
Solution Approach 1:
The invention extracts and amplifies only the specific target sequence of interest from the mixed nucleic acid sample using the specially designed polynucleotide primer. This extraction approach allows subsequent sequencing to focus only on the amplified target region, dramatically reducing the cost while maintaining comprehensive analysis of the disease-specific gene of interest.
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 specific amplification of target nucleic acids, allowing for accurate detection and sequencing of disease-specific genes, improving amplification specificity and rate, and is applicable to various samples including those with mutations or gene variations.
Implementation Method 1
a first region (nucleotides 1 to 15) including a nucleotide sequence complementary to a nucleotide sequence of a portion of a target nucleic acid
Implementation Method 2
a third region (nucleotides 31 to 45) including a nucleotide sequence that self-hybridizes to form a stem-loop structure
Data Source
AI summary
Provided is a polynucleotide including, from the 3′ terminus of the polynucleotide to the 5′ terminus of the polynucleotide, a first region including a nucleotide sequence complementary to a nucleotide sequence of a portion of a target nucleic acid; a second region including a nucleotide sequence identical to a nucleotide sequence of a portion of the target nucleic acid; and a third region including a nucleotide sequence that self-hybridizes to form a stem-loop structure, and compositions, kits, and methods related thereto.