Polymerase Driven NESA for DNA Detection
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Solution Overview
Problem
Current methods for detecting specific DNA sequences, such as rolling circle amplification (RCA) coupled with nicking endonuclease signal amplification (NESA), are slow and prone to background signals due to random circularization, limiting sensitivity and specificity.
Innovation Solution
A method involving an initiating oligonucleotide that forms a complex with the target polynucleotide, extended with a DNA synthesis primer, ligated to create a circular oligonucleotide with a nicking endonuclease recognition site, followed by DNA synthesis and cleavage by a nicking endonuclease, allowing specific detection of the target sequence with reduced false positives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If rolling circle amplification (RCA) is used for DNA amplification coupled with NESA, then sensitivity is improved, but false positive rate increases due to random circularization
Solution Approach 1:
The patent introduces a preliminary action by requiring DNA ligase to repair nicks in the oligonucleotide before RCA can proceed. This preliminary ligation step ensures that only oligonucleotides that have successfully hybridized to the target sequence and been properly ligated can form circles and undergo amplification, thereby preventing random circularization and reducing false positives while maintaining detection sensitivity
Solution Approach 2:
The patent uses DNA ligase as an intermediary component that mediates between the oligonucleotide hybridization and the RCA amplification. The ligase acts as a gatekeeper that must successfully ligate the oligonucleotide ends before amplification can occur, providing an additional layer of specificity that reduces false positives while enabling sensitive detection through subsequent amplification
2Measurement precision
If two-step assay with oligonucleotide circularization is used, then detection capability is improved, but assay time increases
Solution Approach 1:
The patent merges the oligonucleotide circularization step with the DNA amplification step into a single integrated process. The circularized oligonucleotides serve as templates for RCA, combining the specificity of circularization with the sensitivity of amplification in one continuous assay, thereby reducing total assay time while maintaining detection capability
Solution Approach 2:
The patent establishes continuity of useful action by making the circularization process the direct precursor to amplification without intermediate stopping or separate handling steps. The circularized oligonucleotides immediately serve as templates for RCA, maintaining continuous productive action from target binding through amplification, which reduces assay time while preserving detection sensitivity
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 enhances sensitivity and specificity, reducing the false positive rate to less than 10% by introducing a nicking endonuclease recognition site specifically in the circular oligonucleotide, improving the signal-to-noise ratio and confidence in positive results.
Implementation Method 1
exposing the target polynucleotide to an initiating oligonucleotide so that a complex of the target polynucleotide and the initiating oligonucleotide is formed. The complex comprises a first hybridization region formed by the 5′ end sequence and the first hybridization sequence and a second hybridization region formed by the 3′ end sequence and the second hybridization sequence
Implementation Method 2
extending the initiating oligonucleotide in the complex with an extended sequence, which is complementary to the target sequence
Implementation Method 3
ligating the initiating oligonucleotide sequence with the extended sequence to form a circular oligonucleotide
Implementation Method 4
exposing the double stranded DNA to a nicking endonuclease (NE), which can recognize the full NE recognition site and cleave the NE recognition/cutting sequence in the full NE recognition site. The probe in the double stranded DNA is cleaved
Data Source
AI summary
The present invention relates to a novel method for detecting a target polynucleotide having a target sequence, comprising (a) exposing the target polynucleotide to an initiating oligonucleotide; (b) extending the initiating oligonucleotide with an extended sequence complementary to the target sequence; (c) ligating the initiating oligonucleotide sequence with the extended sequence to form a circular oligonucleotide having a nicking endonuclease (NE) recognition/cutting sequence; (d) exposing the circular oligonucleotide to a DNA polymerase and a DNA synthesis primer to synthesize DNA having a NE recognition sequence; (e) exposing the synthesized DNA to a probe having the NE recognition/cutting sequence to form a double stranded DNA having a full NE site; (f) exposing the double stranded DNA to a nicking endonuclease (NE) to cleave the probe; and (g) detecting the cleaved probe. The presence of the cleaved probe indicates the presence of the target polynucleotide.