Circularizing Oligonucleotide Probe Composition for One-Step Variant Detection
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Solution Overview
Problem
Conventional methods for detecting genomic variations using circularizing oligonucleotide probes (COPs) are inefficient with small sample volumes, requiring separate DNA amplification steps and cleanup procedures to eliminate background noise, which are time-consuming and expensive.
Innovation Solution
A reaction condition composition and method that includes DNA ligase, DNA polymerase, circularizing oligonucleotide probes, DNA polymerase buffer, NAD+, primers, and deoxynucleotide triphosphates, allowing for simultaneous hybridization, ligation, and replication of COPs to form circularized probes for genomic variation detection without separate amplification or background noise reduction steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional separate amplification steps are used for small sample volumes, then detection sensitivity is improved, but processing time and complexity increase
Solution Approach 1:
The patent combines hybridization, ligation, and amplification into a single multiplex reaction. The COPs are hybridized to target sequences and simultaneously ligated and amplified in the same reaction mixture without intermediate cleanup steps, resolving the contradiction by integrating multiple sequential operations into one concurrent process that maintains sensitivity while reducing time and complexity
Solution Approach 2:
The reaction composition serves multiple functions simultaneously: it provides hybridization conditions for COP binding, ligation conditions for circularization, and amplification conditions for signal generation. This multi-functional system eliminates the need for separate processing steps while maintaining detection sensitivity for small samples
2Measurement precision
If conventional cleanup procedures are performed to eliminate background noise, then measurement precision is improved, but device complexity and processing time increase
Solution Approach 1:
The patent merges the amplification reaction with the ligation reaction in a single multiplex process. The COPs that successfully hybridize and ligate are amplified simultaneously with the target DNA, while unligated COPs remain in the same reaction mixture but do not interfere with the signal. This eliminates the need for separate cleanup steps to remove background noise while maintaining measurement precision
Solution Approach 2:
The patent accepts the presence of unligated COPs and other background elements in the reaction mixture rather than attempting to remove them. The specific signal from circularized probe amplification distinguishes itself from background noise through its unique amplification characteristics, converting the potential harm of background interference into an acceptable condition that does not require additional cleanup complexity
3Measurement precision
If conventional two-step COP methods are used, then detection accuracy is maintained, but productivity decreases due to multiple sequential steps
Solution Approach 1:
The patent combines the hybridization-ligation step and the amplification step into a single multiplex reaction. The COPs hybridize to target sequences and are simultaneously ligated and amplified in the same reaction tube without intermediate purification. This integration maintains detection accuracy through specific primer binding while dramatically improving productivity by eliminating sequential processing steps
Solution Approach 2:
The patent establishes a continuous reaction process where hybridization, ligation, and amplification occur in an unbroken sequence within the same reaction mixture. The useful action of detecting target DNA continues without interruption or removal from the reaction system, maintaining accuracy through consistent conditions while maximizing throughput through continuous processing
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
Enhances ligation efficiency and detection accuracy of genomic variations in small sample amounts by eliminating the need for additional amplification steps and cleanup procedures, while maintaining high sensitivity and specificity.
Implementation Method 1
The 3′ end is always the —OH group from the last base of the COP and can now ligate to the 5′-end phosphate (P) in the presence of DNA ligase
Implementation Method 2
amplification is accomplished using DNA polymerases that work under isothermal conditions
Implementation Method 3
COPs are selected to hybridize and ligate (i.e. circularize) via DNA ligase to a specific DNA sequence containing a genomic variation (target sequence)
Data Source
AI summary
Reaction condition compositions for detecting a genomic variation from a small sample amount from 5 nano grams (ng) to 1 microgram (ug) includes DNA ligase, DNA polymerase, at least one COP, a DNA polymerase buffer, NAD+, at least two primers, and deoxynucleotide triphosphates (dNTPs). Detection of the genomic variation utilizes COPs with increased ligation efficiency and RCA with fluorescence detection due to simultaneous ligation of COPs to CPs and replication of the genomic variation. The reaction condition composition eliminates the need to perform background reduction of un-hybridized or un-ligated COPs.