Molecular Inversion Probe Assay for Structural Variant Detection
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Solution Overview
Problem
Existing molecular inversion probe (MIP) assays struggle to detect structural variants such as insertions, inversions, translocations, and deletions due to interference from absent or incomplete targeting arm hybridization sites, leading to un-circularized MIPs being discarded and valuable information about structural variants being lost.
Innovation Solution
The method involves using MIPs to detect structural variants by allowing two MIPs to ligate together without circularization, which occurs when a nucleic acid contains a structural variant, and then sequencing the inter-probe product to report the presence of structural variants, while also sequencing circularized MIPs to detect substitutions and small structural variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If MIPs are used to detect mutations, then substitution detection is improved, but structural variant detection capability deteriorates due to absent or incomplete targeting arm hybridization sites
Solution Approach 1:
The invention recovers information that would otherwise be discarded by treating un-circularized MIPs not as waste products to be removed by exonuclease, but as valuable signals containing structural variant information. The un-circularized MIPs are protected from digestion and sequenced to detect structural variants, thereby transforming a previously discarded byproduct into a useful diagnostic signal.
Solution Approach 2:
The MIP assay is enhanced to perform multiple functions simultaneously: circularized MIPs detect substitutions and small indels, while un-circularized MIPs detect structural variants. This multi-functional approach allows a single assay platform to comprehensively screen for different types of genetic mutations without requiring separate specialized methods.
2Reliability
If un-circularized MIPs are discarded to maintain clean assay results, then false positive reduction is improved, but structural variant information is lost
Solution Approach 1:
Instead of discarding un-circularized MIPs as contaminants, the invention recovers and utilizes them as informative signals. By protecting these molecules from exonuclease digestion through phosphorothioate modifications and sequencing them separately, the assay retrieves structural variant information that would otherwise be permanently lost.
Solution Approach 2:
The assay results are segmented into two distinct categories: circularized MIP sequences for substitution detection and un-circularized MIP sequences for structural variant detection. This segmentation allows each type of mutation to be analyzed with appropriate methods while maintaining overall assay reliability and preventing false positives from mixing different signal types.
3Area of stationary object
If MIPs are designed to hybridize to flanking regions, then target coverage is improved, but hybridization efficiency deteriorates when structural variants separate the targeting arms
Solution Approach 1:
The assay dynamically responds to different genomic configurations by producing distinct molecular outcomes: when targeting arms are properly positioned, MIPs circularize; when structural variants separate the arms, MIPs remain un-circularized. This dynamic response allows the system to adapt to various genomic architectures while maintaining reliable detection through appropriate molecular formatting.
Solution Approach 2:
The invention recovers the hybridization signal from MIPs that fail to circularize due to structural variants. By protecting these un-circularized probes from degradation and sequencing them, the assay retrieves valuable information about structural variants that would otherwise be lost, transforming hybridization failure into a productive detection signal.
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 robust and reliable detection and reporting of mutations, including small structural variants and large chromosomal abnormalities, thereby improving the accuracy of carrier screening and genetic analysis.
Implementation Method 1
Each MIP has two linked oligonucleotide targeting arms that are designed to hybridize to a strand of nucleic acid in positions that flank a region of interest
Implementation Method 2
Upon successful hybridization, the two targeting arms of one MIP are connected together in a ligation step into a covalently closed circle
Implementation Method 3
Unbound DNA and un-circularized MIPs are typically digested away by an exonuclease after which the circularized MIPs are used in amplification or sequencing reactions
Implementation Method 4
The inter-probe product can be amplified and/or sequenced. Preferably, the inter-probe product is protected from exonuclease digestion, for example, by including a phosphorothioate base in the MIP backbone
Data Source
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AI summary
The invention relates to carrier screening and methods for describing a structural variant, such as a large rearrangement or chromosomal abnormality, in a person's genome using probes that are designed to determine the person's genetic sequence and reveal substitution mutations and small structural variants. Identifying a structural variant may include exposing a nucleic acid to a plurality of probes. Each probe has a linked pair of targeting arms designed to hybridize upstream and downstream of a target in a genome. The method includes hybridizing two of the probes to the nucleic acid and attaching the two probes together to create an inter-probe product as well as detecting the inter-probe product and reporting a structural variant of the genome in the nucleic acid.