Nuclease-Ligation Nucleic Acid Detection Specificity
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Solution Overview
Problem
Existing nucleic acid detection methods, such as multiplex PCR, LCR, and LDR/PCR, face challenges including non-specific ligations, primer-dimer formation, amplification bias, and high background noise, which limit their ability to specifically amplify single or multiple nucleic acid targets, especially at low frequencies.
Innovation Solution
The method involves using oligonucleotide probe sets with specific configurations, including probes with target-specific portions and overlapping identical nucleotides, which are hybridized to target nucleotide sequences, followed by 5' nuclease activity to cleave the overlapping nucleotide and subsequent ligation to form ligated product sequences, allowing for specific detection of target sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex ligation-based assays are used to detect multiple nucleic acid targets simultaneously, then the throughput and productivity are improved, but non-specific ligations occur generating false positive results
Solution Approach 1:
The assay divides the detection process into distinct functional modules: (1) oligonucleotide probe hybridization to target sequences, (2) selective 5' nuclease cleavage of flap structures, (3) ligation of compatible oligonucleotides, and (4) PCR amplification of ligated products. This segmentation allows each step to be optimized independently, preventing non-specific ligations while maintaining high throughput capability.
Solution Approach 2:
The patent introduces a flap structure as an intermediary element that mediates between the oligonucleotide probes and the target sequence. The flap must be specifically positioned and cleaved by 5' nuclease before ligation can occur, acting as a gatekeeper that prevents non-specific ligation events while enabling specific target detection.
2Adaptability or versatility
If multiple oligonucleotide probes are combined in a single multiplex reaction, then the number of detectable targets increases, but primer-dimer off target complexes form
Solution Approach 1:
The patent applies local quality by designing oligonucleotide probes with position-specific characteristics: the 5' end contains a flap structure with specific sequence composition that is susceptible to 5' nuclease cleavage, while the 3' end contains target-specific binding regions. This local differentiation ensures that only probes with correct local sequences at the junction can undergo ligation, preventing primer-dimer formation even in multiplex reactions.
3Productivity
If traditional PCR amplification is used, then the amplification process is simple and rapid, but amplification bias occurs due to varying primer annealing bias
Solution Approach 1:
The patent performs preliminary action by incorporating the 5' nuclease cleavage step before ligation and amplification. This preliminary cleavage of the flap structure creates a controlled 5' phosphate end that is essential for subsequent ligation, ensuring that only correctly positioned oligonucleotides are amplified, thereby eliminating amplification bias related to varying primer annealing characteristics.
4Reliability
If ligation detection reaction is used to detect nucleic acid sequences, then the detection specificity is improved, but the background noise from abundant 5' phosphate groups increases
Solution Approach 1:
The patent extracts the harmful 5' phosphate groups from the abundant non-specific oligonucleotide probes through selective 5' nuclease cleavage. The nuclease specifically removes the flap structure's 5' end, leaving behind only the necessary 5' phosphate for correct ligation events. This extraction eliminates background noise from non-specific ligation while preserving detection specificity.
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 method enhances the specificity and sensitivity of nucleic acid detection, enabling the identification of low-frequency mutations and rare nucleic acid sequences, such as those found in cancer diagnostics, with reduced background noise and improved multiplexing capabilities.
Implementation Method 1
The overlapping identical nucleotide of the second oligonucleotide probe is cleaved with an enzyme having 5' nuclease activity, thereby liberating a phosphate at the second oligonucleotide probe's 5'end
Implementation Method 2
a ligase is employed to generate a phosphodiester bond across a nick by joining the 5'-phosphate of one oligonucleotide with the 3'-OH of the immediately adjacent oligonucleotide
Implementation Method 3
The first and second oligonucleotide probes of a probe set are configured to hybridize adjacent to one another on the target nucleotide sequence with a junction between the first and second oligonucleotide probes
Data Source
AI summary
The present invention is directed to kits for identifying the presence of one or more target nucleotide sequences in a sample that involve a ligation and/or polymerase reaction. In some embodiments, the ligation products formed in the ligation process of the present invention are subsequently amplified using a polymerase chain reaction. The ligated product sequences or extension products thereof are detected, and the presence of one or more target nucleotide sequences in the sample is identified based on the detection.