Dual-Purpose Primers for Disrupting Nucleic Acid Secondary Structures
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Solution Overview
Problem
Existing nucleic acid hybridization assays face challenges in detecting single nucleotide polymorphisms (SNPs) due to intramolecular secondary structures in target nucleic acids, which prevent efficient hybridization of probes and primers, leading to inconclusive results, especially in regions like exons 1 and 2 of the cytochrome P450 CYP2D6 gene.
Innovation Solution
A dual-purpose primer is introduced, which includes a primer sequence for amplifying target nucleotide sequences and a blocking sequence to disrupt secondary structure formation, allowing for effective hybridization and detection of SNPs. This primer can be substantially shorter than previous blocking probes and does not require perfect complementarity with the target sequence, facilitating access to masked sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional primers are used for amplification, then amplification can proceed, but secondary structure formation in the target nucleic acid blocks probe hybridization and prevents SNP detection
Solution Approach 1:
The patent combines two previously separate functions into a single primer: (1) the primer function for amplifying target DNA, and (2) the blocking probe function for disrupting secondary structures. This merged dual-purpose primer simultaneously performs amplification and secondary structure disruption, eliminating the need for separate blocking probes and improving SNP detection reliability in regions with secondary structures
Solution Approach 2:
The primer is designed with multi-functionality, serving both as an amplification primer and as a blocking agent. By incorporating a blocking sequence that is substantially complementary to the secondary structure-forming region, the primer universally addresses both amplification needs and secondary structure interference in a single molecular entity
2Reliability
If external blocking probes are used to disrupt secondary structures, then probe hybridization improves, but the system requires multiple separate components increasing complexity
Solution Approach 1:
The patent merges the blocking probe and amplification primer into a single dual-purpose primer molecule. This eliminates the need for separate external blocking probes and simplifies the assay system from multiple components to a unified primer that performs both functions simultaneously
3Reliability
If blocking probes are used to prevent secondary structure formation, then hybridization is improved, but the blocking probes must be substantially complementary to the target sequence requiring longer length
Solution Approach 1:
The dual-purpose primer applies local quality by having different regions serve different functions: the 3' end contains the primer sequence for amplification while the 5' end contains the blocking sequence that is substantially complementary to the secondary structure-forming region. This localized functional differentiation allows the blocking sequence to be shorter than traditional blocking probes while maintaining effectiveness
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 dual-purpose primer system significantly enhances the detection signal for SNPs by disrupting unwanted secondary structures, enabling accurate amplification and genotyping, with a 100-fold increase in signal compared to conventional methods and a 5- to 7-fold increase over methods using external blocking probes, as demonstrated in experiments with the cytochrome P450 CYP2D6 gene.
Implementation Method 1
Hybridization is based upon pairing between complementary nucleic acid strands. The blocking sequence hybridizes to a region of the target nucleic acid that would otherwise form an intramolecular secondary structure.
Implementation Method 2
Intramolecular secondary structures in a single-stranded nucleic acid arise from the intramolecular formation of hydrogen bonds between complementary nucleotide sequences within the single-stranded nucleic acid itself.
Implementation Method 3
The primer sequence is effective to amplify a target nucleotide sequence in a target molecule
Implementation Method 4
a fluorophore and a quencher are in self-quenching proximity. Upon denaturation and hybridization with a target nucleotide sequence, however, the segment of the probe that was formerly in the hairpin structure hybridizes with the amplified target sequence, and is detectable by the increased fluorescence.
Data Source
AI summary
The present invention provides primers and probes to be used in a method of enhancing hybridization of a probe to a target nucleotide sequence when the target sequence is capable of forming intramolecular secondary structures that interfere with hybridization of the probe to the target sequence. In particular, the invention includes a primer for amplifying a target nucleotide sequence, wherein at least a portion of the target nucleotide sequence can form an intramolecular secondary structure. The primer of the invention includes a primer nucleotide sequence complementary to a portion of the target nucleotide sequence that does not form a secondary structure, and a blocking sequence substantially complementary to at least a portion of the secondary structure-forming region of the amplified target nucleotide sequence, wherein the blocking sequence hybridizes to a portion of the secondary structure-forming region of the amplified target nucleotide sequence and blocks the formation of the secondary structure.