Oligonucleotide Probe Universal Base Silent Mutation Suppression
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Solution Overview
Problem
Current genomic analysis methods are unable to distinguish between clinically significant and nonsignificant mutations, often identifying phenotypically neutral or silent mutations alongside important ones, due to their sensitivity to all types of mutations.
Innovation Solution
A method using oligonucleotide probes with specific melting temperatures (Tm) for wild and variant alleles, incorporating universal bases at sites where nonsignificant mutations are not desired, allowing for differential detection of variants by hybridization and PCR amplification, while suppressing detection of silent mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional oligonucleotide probes are used to detect all mutations, then sensitivity to detect variant alleles is improved, but nonsignificant silent mutations are also detected causing false positives
Solution Approach 1:
The probe is designed with differentiated local properties: positions 1-7 are designed to specifically detect clinically significant mutations through strict base pairing, while position 8 uses a universal base (inosine) that forms stable pairs with all four DNA bases, thereby suppressing detection of silent mutations at that position. This local differentiation allows the probe to distinguish between significant and nonsignificant variants.
Solution Approach 2:
The invention changes the chemical parameter of the nucleotide at position 8 from a specific base (A, T, C, or G) to a universal base (inosine). This parameter change alters the hybridization behavior, allowing the probe to maintain stable binding regardless of the base present at that position, thereby suppressing detection of silent mutations while preserving detection of clinically significant mutations elsewhere in the probe sequence.
2Adaptability or versatility
If the probe sequence is designed to detect multiple variants, then versatility is improved, but the ability to distinguish clinically significant from nonsignificant mutations deteriorates
Solution Approach 1:
The probe employs local quality differentiation where positions 1-7 are designed with specific base requirements to detect clinically significant mutations, while position 8 uses a universal base (inosine) that accepts any base pairing. This creates localized detection zones with different specificity levels, allowing the single probe to versatilely detect multiple clinically significant variants while suppressing nonsignificant silent mutations.
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
Enables sensitive detection of clinically significant mutations while avoiding the identification of phenotypically neutral mutations, allowing for precise analysis of genomic sequences.
Implementation Method 1
allowing the probe to hybridise to a target nucleic acid sequence
Implementation Method 2
determining the Tm of the probe when hybridised to the target nucleic acid sequence
Data Source
AI summary
A method and an oligonucleotide probe are described for determining the presence or absence of mutant alleles in a genomic locus. The probe binds to different alleles of a target sequence with different melting temperatures (Tm). The method determines the Tm of the probe when it is hybridized to the target sequence to establish whether a variant nucleic acid such as a mutant allele is present or absent in the target sequence. There may be variants in a target sequence that are not of interest, for example phenotypically silent mutations. To ensure that these variants do not influence the Tm of the probe, the probe contains universal base sites where such variants of no interest occur.