Nucleic Acid Probe Design for Specific Detection
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Solution Overview
Problem
Current methods for detecting target nucleic acid sequences in biological samples are inefficient due to the requirement for stem-loop structures between anchor and reporter domains, which decreases probe binding to the target in the absence of the nucleic acid, and are extendible by polymerases, limiting specificity and accuracy.
Innovation Solution
A two-domain probe system with an anchor domain that drives hybridization and a reporter domain linked by a non-nucleoside linker, where neither domain forms a stem loop without the target nucleic acid, and is designed to be non-extendible, allowing for specific detection of target sequences through changes in melting temperature or fluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If stem-loop structures are formed between anchor and reporter domains, then probe stability is improved, but probe binding to target nucleic acid decreases
Solution Approach 1:
The invention extracts the stem-loop structure from the probe design, eliminating the intramolecular hybridization between anchor and reporter domains. By removing this self-complementary structure, the probe avoids forming stable stem-loops that would reduce target binding efficiency, while maintaining probe stability through alternative means such as appropriate domain composition and length.
Solution Approach 2:
The probe is segmented into distinct anchor and reporter domains connected by a flexible linker, allowing each domain to function independently. The anchor domain binds to the target nucleic acid while the reporter domain remains available for detection, preventing the formation of intramolecular stem-loops that would compromise target binding.
2Adaptability or versatility
If probe is extendible by polymerase, then amplification capability is improved, but detection specificity decreases
Solution Approach 1:
The probe incorporates a localized modification at the 3' end (such as a dideoxynucleotide or inverted dT) that specifically prevents polymerase extension only at this critical position. This local quality change maintains the probe's ability to bind specifically to the target sequence while eliminating unwanted amplification, thereby preserving detection specificity.
3Stability of the object's composition
If anchor and reporter domains are highly complementary, then probe stability is improved, but detection accuracy decreases
Solution Approach 1:
The probe design applies local quality by making only the reporter domain highly complementary to the target sequence, while the anchor domain has lower complementarity. This localized high complementarity in the reporter region enables accurate detection of the target sequence without requiring the entire probe to be highly complementary, thus maintaining detection accuracy while achieving sufficient stability.
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 highly specific detection of target sequences, including SNPs and mutations, with improved accuracy and specificity, distinguishing between matches and mismatches, and is suitable for various applications such as molecular diagnostics and pharmacogenomics.
Implementation Method 1
the hybridization between the anchor domain and the target nucleic acid by itself is sufficient to drive the hybridization of the probe onto the intended location of the target nucleic acid
Implementation Method 2
the detecting step comprises measuring the melting temperature of a complex formed between the reporter domain and the target nucleic acid
Implementation Method 3
the method further comprises contacting the probe and sample with a soluble intercalating quencher such that the quencher alters fluorescence from the label when the reporter domain forms a complex with the target nucleic acid
Data Source
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AI summary
The invention provides, inter alia, novel probes, methods, reaction mixtures, and kits for detecting the presence or absence of a target nucleic acid sequence.