Hairpin Probe Amplification for Multiplex Genotyping
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genotyping methods, such as multiplex PCR, require substantial upfront investment and are limited by primer dimer issues, making them inefficient and costly for identifying DNA and RNA sequences with target nucleotide sequences.
Innovation Solution
The use of hairpin formation in nucleotide sequences, promoted by complementary probe sequences, allows for selective amplification of target regions through polymerase chain reaction, reducing the need for multiple primers and minimizing primer dimer formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiplex PCR is used to enrich DNA for multiple loci, then the ability to identify multiple genomic markers is improved, but the device complexity and upfront investment increase substantially
Solution Approach 1:
The invention divides the complex multiplex PCR system into simpler components by using hairpin-forming oligonucleotides that can be individually designed for each genomic marker. Each hairpin oligonucleotide contains a target-specific region and a universal region, allowing separate design and assembly of multiple markers without requiring complex primer combinations.
Solution Approach 2:
The invention creates a universal hairpin oligonucleotide structure that can bind to multiple different target sequences through the target-specific region while maintaining a common universal region for standardized processing. This universal design allows the same basic mechanism to be applied across multiple genomic markers, reducing the need for marker-specific complex procedures.
2Adaptability or versatility
If multiplex PCR is used to enrich DNA for multiple loci, then the ability to identify multiple genomic markers is improved, but the loss of time and upfront investment increase
Solution Approach 1:
The invention performs preliminary design of hairpin oligonucleotides with pre-defined target-specific and universal regions. This preliminary structuring allows rapid assembly of new marker panels without requiring extensive optimization time, as the basic hairpin framework is already established and can be quickly adapted to new targets.
Solution Approach 2:
The invention enables rapid adjustment of detection parameters by simply changing the target-specific region sequence of the hairpin oligonucleotides while maintaining the same universal processing conditions. This parameter flexibility allows quick adaptation to different genomic markers without requiring extensive re-optimization of PCR conditions or protocols.
3Quantity of substance
If multiple primer pairs are used in multiplex PCR, then coverage of multiple loci is improved, but primer dimer formation increases reducing amplification efficiency
Solution Approach 1:
Instead of using traditional forward and reverse primers that can anneal to each other forming dimers, the invention inverts the approach by using hairpin-forming oligonucleotides where the complementarity is built into the single molecule structure. The hairpin forms intramolecularly rather than requiring intermolecular primer-primer annealing, eliminating the primer dimer problem.
Solution Approach 2:
The invention introduces a hairpin structure as an intermediary between the target DNA and the amplification process. The hairpin oligonucleotide first binds to the target sequence, forming a stable hybrid, and then serves as the template for amplification. This intermediary step prevents direct primer-primer interactions that lead to dimer formation.
4Adaptability or versatility
If traditional genotyping methods are used, then genomic markers can be evaluated, but hands-on effort and costs increase
Solution Approach 1:
The hairpin oligonucleotides are designed to self-fold into the hairpin structure under the assay conditions, eliminating the need for complex assembly steps or specialized equipment. The target-specific and universal regions automatically organize into the functional hairpin conformation when exposed to the target DNA, reducing hands-on manipulation requirements.
Solution Approach 2:
The invention combines the target recognition function and the amplification template function into a single hairpin oligonucleotide molecule. This merging of functions eliminates the need for separate probe and primer components, simplifying the assay protocol and reducing the number of steps requiring manual intervention.
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 reduces upfront costs and time, enabling rapid and flexible genotyping with reduced hands-on effort, while enriching genomic DNA for downstream sequencing and allowing evaluation of various genomic markers, including SNPs, STRs, insertions, and deletions.
Implementation Method 1
the probe nucleotide sequence being complementary to the target region nucleotide sequence
Implementation Method 2
Amplifying may include a polymerase chain reaction
Data Source
AI summary
DNA or RNA including a target nucleotide sequence are amplified by placing a probe nucleotide sequence on the DNA or RNA. The probe nucleotide sequence is complementary to the target nucleotide sequence. The interaction between the probe nucleotide sequence and target nucleotide sequence causes the single-stranded DNA to form a hairpin. The hairpinned single-stranded DNA are selected for amplification.