VD-PTOCE Assay for Minority Mutation Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods for detecting nucleotide variations, such as SNPs, face challenges including false positive results due to non-specific hybridization, limitations in multiplex target detection, and difficulties in allelic-specific probe design and optimized reaction conditions, particularly in clinical samples with low-abundance mutant alleles.
Innovation Solution
The development of a VD-PTOCE assay using an amplification blocker and a PTO-NV (Probing and Tagging Oligonucleotide for Nucleotide Variation) that selectively amplifies target nucleotide variations by employing a nucleotide variation discrimination site and resisting 5' nuclease cleavage, allowing for accurate detection of low-abundance mutant alleles without amplifying wild-type alleles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional hybridization-based methods are used for nucleotide variation detection, then the detection process is simple, but false positive results occur due to non-specific hybridization between probes and non-target sequences
Solution Approach 1:
The assay is divided into distinct functional components: an upstream primer for amplification, a PTO-NV probe for specific binding and signal generation, and a downstream primer for amplification. This segmentation allows each component to perform its specific function, reducing non-specific hybridization while maintaining assay simplicity
Solution Approach 2:
The PTO-NV probe acts as an intermediary between the target sequence and the detection system. It specifically binds to the target nucleotide variation and generates a detectable signal through 5' nuclease cleavage, providing reliable detection while preventing false positives from non-specific hybridization
2Measurement precision
If TaqMan probe method with polymerization-dependent cleavage is used, then signal generation is achieved, but the upstream primer must be extended before the polymerase can cleave the probe, adding reaction time
Solution Approach 1:
The PTO-NV probe is designed with a 5' tail region that is pre-positioned and non-hybridizable with the target sequence. This preliminary configuration allows the polymerase to immediately cleave the probe upon binding, eliminating the need for prior primer extension and reducing reaction time while maintaining signal detection accuracy
3Measurement precision
If amplification is performed on clinical samples with low-abundance mutant alleles, then the mutant alleles can be detected, but wild-type alleles are also amplified, overwhelming the signal from mutant alleles
Solution Approach 1:
The PTO-NV probe is designed with a nucleotide variation discrimination site that specifically recognizes the mutant allele sequence. This local specificity ensures that the probe binds only to mutant alleles and generates signals only from mutant amplification products, eliminating interference from wild-type allele amplification while maintaining high sensitivity for low-abundance mutant detection
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 reliable and reproducible detection of multiple nucleotide variations in low-abundance samples, improving accuracy and convenience by preventing wild-type allele amplification and allowing for specific detection of target nucleotide variations, even in excess of wild-type alleles.
Implementation Method 1
DNA hybridization is a fundamental process in molecular biology and is affected by ionic strength, base composition, length of fragment to which the nucleic acid has been reduced, the degree of mismatching, and the presence of denaturing agents.
Implementation Method 2
In TaqMan probe method, the labeled probe hybridized with a target nucleic acid sequence is cleaved by a 5' nuclease activity of an upstream primer-dependent DNA polymerase, generating a signal indicating the presence of a target sequence
Implementation Method 3
In polymerization-dependent cleavage, extension of the upstream primer must occur before a nucleic acid polymerase encounters the 5'-end of the labeled probe. As the extension reaction continues, the polymerase progressively cleaves the 5'-end of the labeled probe.
Data Source
AI summary
The present invention relates to the detection of a nucleotide variation on a target nucleic acid sequence using an amplification blocker and a VD-PTOCE (Variation Detection by PTO Cleavage and Extension) assay. The present invention is significantly effective in the detection of a minority mutation in an excess of wild-type DNA.